Immunogenic compositions and vaccines for preventing or treating filarial disease

ABSTRACT

The present disclosure is directed to an immunogenic composition including: at least one or at least two isolated polypeptides or immunogenic fragments thereof, and optionally a pharmaceutically acceptable carrier, wherein each polypeptide is expressed on a luminal surface of an intestine of a filarial worm, wherein each polypeptide is expressed at a level at least two-fold higher in the intestine in comparison to the level of expression of each polypeptide in a reproductive tract or a body wall of the filarial worm, wherein each isolated polypeptide has at least one transmembrane domain, and wherein each polypeptide is a non-mitochondrial polypeptide. Also provided herein is a method for preventing or treating a filarial disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage application ofPCT/US2016/022787 filed Mar. 17, 2016, which claims the benefit of, andrelies on the filing date of, U.S. provisional patent application No.62/135,115, filed Mar. 18, 2015, the entire disclosure of which isincorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under grant numberR073UE awarded by the Uniformed Services University. The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 9, 2016, isnamed HMJ-150-PCT_ST25.txt and is 120,000 bytes in size.

BACKGROUND

The filariae are thread-like parasitic nematodes (roundworms) that aretransmitted by arthropod vectors. The adult worms inhabit specifictissues where they mate and produce microfilariae, the characteristictiny, thread-like larvae.

The microfilariae infect vector arthropods, in which they mature toinfective larvae. Diseases caused by filariae are a major health problemin many tropical and subtropical areas. Wuchereria bancrofti and Brugiamalayi are filarial parasites that are the major causative agents oflymphatic filariasis. Currently, it is estimated that over 129 millionpeople are infected with either of these organisms and over one billionlive in at-risk areas. Since 2000, there has been an ongoing effortthrough the Global Program to Eliminate Lymphatic Filariasis toeradicate these infections. While this program is having a substantiveimpact on the prevalence of infection, its efficacy is limited by theneed to repeatedly treat entire endemic populations for 6-10 years. Theadvent of new tools, such as vaccines or more effective anthelmintics,would be of great benefit toward these eradication efforts.

One of the principle obstacles in designing vaccines against suchparasitic worms, however, is that previously exposed individualsfrequently have IgE antibodies to surface and secreted worm antigens,putting them at risk for allergic reactions when re-exposed to theseantigens. Since intestinal antigens of helminths may be “hidden” fromthe immune response during natural infection, yet accessible byantibodies after antigen administration, homogenates of such antigenshave been proposed for use in vaccines. However, while the genomes ofWuchereria bancrofti and Brugia malayi, as well as the filarial genomesof the causative agents of loiasis and river blindness have beencompleted, the anatomic localization of proteins in these filarial wormsis unknown. Moreover, the use of homogenates from helminth intestineshas resulted in variable efficacy.

Other diseases known in the art, which are caused by filarial parasites,include heartworm disease. This disease is caused by the parasiteDirofilaria immitis. The physical presence of the heartworm parasite inthe pulmonary artery and right ventricle of the canine heart, forexample, and the resulting destruction of tissue, causes respiratory andcirculatory problems which can be fatal under conditions of stress orvigorous exercise. The heartworm parasite has also been shown to be thecause of focal lung, liver, eye and cutaneous lesions in man. Currently,heartworm disease is treated by administering anti-parasitic agents toinfected animals. Unfortunately, heartworm disease that has not beendiagnosed in its early stages may be quite refractile to treatment.

Accordingly, there is a need in the art for immunogenic compositions,such as vaccines, which may be used to prevent or treat diseases causedby filarial worms including lymphatic filariasis, loiasis, riverblindness and heartworm.

BRIEF SUMMARY

Further areas of applicability of the present disclosure will becomeapparent from the detailed description provided hereinafter. It shouldbe understood that the detailed description and specific examples, whileindicating embodiments of the disclosure, are intended for purposes ofillustration only and are not intended to limit the scope of thedisclosure.

The present disclosure is directed to an immunogenic compositionincluding: at least one or at least two isolated polypeptides orimmunogenic fragments thereof, and optionally a pharmaceuticallyacceptable carrier, wherein each polypeptide is expressed on a luminalsurface of an intestine of a filarial worm, wherein each polypeptide isexpressed at a level at least two-fold higher in the intestine incomparison to the level of expression of each polypeptide in areproductive tract or a body wall of the filarial worm, wherein eachisolated polypeptide has at least one transmembrane domain, and whereineach polypeptide is a non-mitochondrial polypeptide.

Also provided herein is a method for preventing or treating a filarialdisease including administering an effective amount of a vaccinecomposition including at least one or at least two isolated polypeptidesor immunogenic fragments thereof to a subject in need thereof, whereineach polypeptide is expressed on a luminal surface of an intestine of afilarial worm, wherein each polypeptide is expressed at a level at leasttwo-fold higher in the intestine in comparison to the level ofexpression of each polypeptide in a reproductive tract or a body wall ofthe filarial worm, wherein each isolated polypeptide has at least onetransmembrane domain, and wherein each polypeptide is anon-mitochondrial polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will become more fully understood from thedetailed description and the accompanying drawings, wherein:

FIG. 1 depicts the anatomy of adult female B. malayi. The tissues andstructures dissected for the proteomic analysis as described in theExamples include the body wall, reproductive tract and intestine.

FIGS. 2A and 2B depict the dissection process of adult female B. malayi.FIG. 2A shows a break in the body wall and extrusion of the gut andreproductive tract as described in the Examples. Magnification: topleft: 40×, bottom left: 100×. FIG. 2B shows the body wall in the processof being slid away from the gut and reproductive tract. Magnification:top right: 30×, bottom right: 20×.

FIG. 3A depicts a Venn diagram of proteins identified within eachanatomic fraction of adult female Brugia malayi based on a 2 peptideminimum for identification as described in the Examples.

FIG. 3B depicts a heat map of hierarchical clustered proteins foundwithin the different anatomic fractions of adult female B. malayi.

FIG. 4 depicts the association of transporter proteins with theintestine as measured by gene set enrichment analysis (GSEA)P-value=0.005, as described in the Examples.

FIG. 5A depicts the association of immunological (P-value=0.003)proteins with the body wall of the adult female B. malayi as measured byGSEA, as described in the Examples.

FIG. 5B depicts the association of cytoskeletal proteins (P-value=00.009) with the body wall of the adult female B. malayi as measured byGSEA, as described in the Examples.

FIG. 6A depicts the association of transcription (P<0.001 with thereproductive tract of adult female B. malayi as described in theExamples.

FIG. 6B depicts the association of nuclear regulation proteins (P=0.013)with the reproductive tract of adult female B. malayi as described inthe Examples.

DETAILED DESCRIPTION

The following description of the embodiments is merely exemplary innature and is in no way intended to limit the disclosure, itsapplication, or uses.

As used throughout, ranges are used as shorthand for describing each andevery value that is within the range. Any value within the range can beselected as the terminus of the range. In addition, all references citedherein are hereby incorporated by reference in their entireties. In theevent of a conflict in a definition in the present disclosure and thatof a cited reference, the present disclosure controls.

Unless otherwise specified, all percentages and amounts expressed hereinand elsewhere in the specification should be understood to refer topercentages by weight. The amounts given are based on the active weightof the material.

Immunogenic Composition

In some embodiments, the present disclosure encompasses an immunogeniccomposition comprising at least one isolated polypeptide as describedherein or an immunogenic fragment thereof and optionally apharmaceutically acceptable carrier.

The term “polypeptide” as used herein refers to a polymer of amino acidresidues. This term is used interchangeably with the term “protein.”

In some embodiments, the present polypeptides are obtained or derivedfrom a filarial worm. A “filarial worm” as used herein refers toparasitic nematodes of the Metazoa kingdom including the superfamilyfilarioidea, family Filariidae. Filarial worms include, but are notlimited to, species belonging to the genera Brugia, such Brugia malyai,Wuchereria, such as Wuchereria bancrofti, Onchocerca, such as Onchocercavolvulus, Loa, such as Loa loa and Dirofilaria, such as Dirofilariaimmitis. The phrase “derived from” encompasses actually or theoretically“originating from,” “obtained from,” or “isolated from” a parentpolypeptide.

In some embodiments, the polypeptides of the present immunogenic areexpressed on a luminal surface of a filarial worm intestine. Withoutbeing bound by theory, it is believed that administering such intestinalluminal surface polypeptides to a subject may mitigate the possibilityof an allergic reaction in the subject. Because intestinal antigens maybe “hidden” from the immune response during natural infection, yetaccessible by antibodies induced by administration, intestinal antigensare likely to have a low potential for eliciting allergic responses whenadministering, such as vaccinating, previously infected individuals.Further, it is believed that administering, such as vaccinating, asubject with particular intestinal antigens, such as the polypeptides orimmunogenic fragments thereof described herein, may be more effectivethan vaccinating with crude homogenates of antigens since, for example,sufficient amounts of effective antigen may not be present in suchmixtures. Accordingly, in some embodiments, the present immunogeniccomposition consists essentially of the isolated polypeptides disclosedherein, such as one or more isolated polypeptides selected from SEQ IDNOS: 1-27 or immunogenic fragments thereof and a pharmaceuticallyacceptable carrier and does not encompass crude homogenates of antigen,such as a crude homogenate of intestinal proteins.

In various embodiments, the present polypeptides are enriched in theintestine of a filarial worm in comparison to another anatomic fractionof a filarial worm. As used herein, “enriched” or “abundant” refers to apolypeptide that is naturally found to be expressed at a higher level inthe intestine, for example, in comparison to other anatomic fractions,such as the reproductive tract or the body wall. See FIG. 1, whichdepicts the anatomy of a filarial worm. In some embodiments, theexpression level of the polypeptide is at least two fold higher, such asat least three fold higher, such as at least five fold higher, such asat least 10 fold higher, such as at least 50 fold higher in theintestine in comparison to the expression level of the polypeptide inthe reproductive tract or the body wall of the filarial worm.

Enrichment or abundance of polypeptides can be assessed by any methodknown in the art. For example, protein separation and comparison bytwo-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followedby mass spectrometry (MS) or tandem mass spectrometry (MS/MS)identification may be used for quantitative analysis of proteinmixtures, see, for example, H. J. Issaq and T. D. Veenstra,BioTechniques, vol. 44, no. 5, pp. 697-700, 2008, which is hereinincorporated by reference. In this method, the intensity of the proteinstain is used to make a determination regarding the quantity of aparticular protein.

Protein quantitation may also be assessed using non-gel-based “shotgun”proteomic techniques such as Multidimensional Protein Identification(MudPIT). See e.g., A. Motoyama and J. R. Yates III, AnalyticalChemistry, vol. 80, no. 19, pp. 7187-7193, 2008 and B. Domon and R.Aebersold, Science, vol. 312, no. 5771, pp. 212-217, 2006, which areeach herein incorporated by reference.

Non-gel based proteomic methods may include (i) sample preparationincluding protein extraction, reduction, alkylation, and digestion; (ii)sample separation by liquid chromatography (LC or LC/LC) and analysis byMS/MS; (iii) data analysis including peptide/protein identification,quantification, and statistical analysis. For example, each sample maybe separately prepared, then subjected to individual LC-MS/MS orLC/LC-MS/MS runs. Protein abundance may be assessed, for example, usingspectral counting of identified proteins after MS/MS analysis. Spectralcount may be measured for individual LC-MS/MS or LC/LC-MS/MS runs andchanges in protein abundance may be calculated via a direct comparisonbetween different analyses.

Typically, normalization and statistical analysis of spectral countingdatasets are used to detect changes in protein abundance in complexmixtures. See, for example, McIlwain et al., 2012, BMC Bioinformatics,13, 308, Paoletti et al., 2006, Proc. Natl. Acad. Sci. U.S.A., 103,18928-18933, regarding determining the relative abundance of a singleprotein between samples and Liu et al., 2004, Anal. Chem., 76,4193-4201, for example, regarding estimating relative abundance betweendifferent proteins in one sample. These references are hereinincorporated in their entireties.

The relative abundance of the present polypeptides may be assessed usingnormalization and statistical analysis of spectral counting datasets asdescribed in the present Examples. For example, Normalized SpectralAbundance Factor (NSAF) and NSAF enrichment values may be determined(see Examples). In some embodiments, the polypeptides of the presentdisclosure exhibit an NSAF enrichment value of at least 2, such as atleast 3, such as at least 4, such as at least 20 in the intestine andless than 2, such as less than 1 or less than 0.5 in other atomicfractions, such as the body wall or the reproductive tract.

In some embodiments, the polypeptides of the present immunogeniccomposition have at least one transmembrane domain, such as 1-2transmembrane domains, to facilitate recombinant expression and anon-cytoplasmic portion to increase the likelihood of interaction withantibodies ingested by filariae. The non-cytoplasmic portion may be forexample, 50 amino acids in length or more, such as 100 amino acids inlength or more, such as 500 amino acids in length or more. Such featuresmay be determined using methods known in the art or predicted using artknown software, e.g. Interpro, Zdobnov E M, Apweiler R. 2001.InterProScan-an integration platform for the signature-recognitionmethods in InterPro. Bioinformatics 17: 847-848, which is hereinincorporated by reference.

In some embodiments, the polypeptide of the present disclosure isspecific to the intestine. “Specific” in reference to a particularanatomical fraction of the worm means that the polypeptide was onlyidentified within the specific fraction, e.g., the intestine.

In some embodiments, the present polypeptides are non-mitochondrialpeptides, i.e. not present, targeted to or expressed in themitochondria.

As noted above, the present composition comprising at least one isolatedpolypeptide is an immunogenic composition. As used herein, the term“immunogen” or “immunogenic” refers to any substrate that elicits animmune response in a host, e.g., at least an antibody response. An“immunogenic composition” includes at least one isolated polypeptidewith or without a pharmaceutically acceptable carrier, such as anadjuvant. The immunogenic compositions disclosed herein may or may notbe immunoprotective or therapeutic. Accordingly, the term “immunogenic”is not intended to be limited to vaccines.

In some embodiments, the immunogenic composition of the presentdisclosure is a vaccine. As used herein, a vaccine encompasses animmunogenic composition that prevents, ameliorates, palliates, oreliminates disease from a host, such as the diseases described herein.

In other embodiments, the immunogenic composition described herein maybe used to obtain an antibody composition, which may then beadministered to a subject to provide temporary immunity, i.e.,artificially acquired passive immunity. Methods for preparing andadministering such antibody compositions are known in the art and aredescribed, for example, in U.S. Pat. No. 4,748,018, which is hereinincorporated by reference in its entirety.

As described herein, a polypeptide of the present immunogeniccomposition is an isolated polypeptide. The terms “isolated” or“purified” or “biologically pure” refer to material that issubstantially or essentially free from components that normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is considered to be substantially purified. The term“purified” denotes that a protein gives rise to essentially one band inan electrophoretic gel. For example, it means that the protein is atleast 85% pure, such as at least 95% pure or at least 99% pure.

In some embodiments, the polypeptide of the present immunogeniccomposition is a polypeptide selected from SEQ ID NOS: 1-27. In someembodiments, the isolated polypeptide is a cell adhesion protein, suchas a polypeptide of SEQ ID NOS: 1 or 2. In some embodiments, theisolated polypeptide is a cell signaling protein, such as a polypeptideof SEQ ID NOS: 3 or 4. In some embodiments, the polypeptide is achaperone/HSP protein, such as a polypeptide of SEQ ID NOS: 5 or 6. Insome embodiments, the polypeptide is involved in xenobiotic metabolismusing glucuronidation, for example, a UDP-glucuronosyl or a UDP glucosyltransferase, such as a polypeptide of SEQ ID NOS: 7 or 8, respectively.In some embodiments, the isolated polypeptide is a protease such as SEQID NOS: 13, 14 or 15. In some embodiments, the isolated polypeptide is aprotease inhibitor, such as SEQ ID NO: 17. A description of SEQ ID NOS:1-27 is found in Table 7 and Tables C and D.

In some embodiments, the polypeptide of the present immunogeniccomposition is an immunogenic fragment, such as an immunogenic fragmentof SEQ ID NOS: 1-27. The phrase “immunogenic fragment” or “immunogenicportion” refers to a fragment or truncated form of an amino acidsequence, for example, an amino acid sequence selected from the groupconsisting of SEQ ID NOS. 1-27 that elicits an immunological response.

In some embodiments, the polypeptide of the present immunogeniccomposition is an immunogenic variant, such as an immunogenic variant ofSEQ ID NOS: 1-27. The phrase “immunogenic variant” refers to asubstituted form of an amino acid sequence, for example, an amino acidsequence selected from the group consisting of SEQ ID NOS. 1-27 thatelicits an immunological response. Insertions and/or deletions may alsobe incorporated into an immunogenic variant.

In some embodiments, the immunogenic variant of the present disclosurecomprises a substitution, such as a conservative substitution forexample, a substituted form of an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 1-27 containing one or more conservativeamino acid substitutions. A “conservative amino acid substitution” isone in which the amino acid residue is replaced with an amino acidresidue having a side chain with a similar charge. Families of aminoacid residues having side chains with similar charges have been definedin the art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

In general, the immunogenic fragments and variants described herein,such as fragments or variants of the polypeptides of SEQ ID NOS: 1-27comprise at least one epitope and include at least six contiguous aminoacids from the full-length protein, e.g., at least six contiguous aminoacids from the cell adhesion protein set forth in SEQ ID NO: 1. Moretypically, the present variants or fragments will have at least 10, evenmore typically at least 15, and still more typically at least 19, andyet even more typically 30 contiguous amino acids from the full-lengthprotein, e.g., the cell adhesion protein set forth in SEQ ID NO: 1.

The term “epitope” means a segment or fragment of a composition ofmatter, e.g., a polypeptide, which is recognized by the immune system,specifically by antibodies, B cells, or T cells. In some embodiments,the epitope is generally a fragment or fragments of a polypeptide setforth in SEQ ID NOS: 1-27.

Such fragments can be identified using any number of epitope mappingtechniques, well known in the art. See, e.g., Epitope Mapping Protocolsin Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996)Humana Press, Totowa, N.J), which is herein incorporated by reference.For example, linear epitopes may be determined by concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of a protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known and described in the art, see e.g.,U.S. Pat. No. 4,708,871; Geysen et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81, 3998-4002; and Geysen et al., 1986, Molec. Immunol.23,709-715, which are herein incorporated by reference in theirentireties. Similarly, conformational epitopes are readily identified bydetermining spatial conformation of amino acids, such as by, e.g., x-raycrystallography and two-dimensional nuclear magnetic resonance. SeeEpitope Mapping Protocols, supra.

In some embodiments, the polypeptides of the present disclosureencompass polypeptides that are substantially homologous to thepolypeptides set forth in SEQ ID NOS: 1-27. The substantially homologouspolypeptides may be from or derived from any filarial species or generaincluding but not limited to Brugia, such as Brugia malyai, Wuchereria,such as Wuchereria bancrofti, Onchocerca, such as Onchocerca volvulus,Loa, such as Loa loa and Dirofilaria, such as Dirofilaria immitis. Insome embodiments, the polypeptides of the present disclosure, which aresubstantially homologous to the polypeptides set forth in SEQ ID NOS:1-27 are not substantially homologous to a human polypeptide.

In some embodiments, the immunogenic composition includes at least oneisolated polypeptide comprising an amino acid sequence selected from SEQID NOS: 1-7, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NOS: 18-20, SEQ ID NO:22, SEQ ID NO: 23 and SEQ ID NO: 26.

In specific embodiments, the immunogenic composition includes at leastone, for example, polypeptide obtained Dirofilaria immitis anddisplaying substantial homology to a polypeptide selected from SEQ IDNOS: 1-7, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NOS: 18-20, SEQ ID NO:22, SEQ ID NO: 23 and SEQ ID NO: 26.

As used herein, “homology” refers to the percent sequence identitybetween two polypeptide moieties. Two polypeptide sequences “displaysubstantial homology” or are “substantially homologous” to each otherwhen the sequences exhibit at least about 41%, such as at least about75%, more typically at least about 80%-85%, even more typically at leastabout 90%, and most typically at least about 95%, 96%, 97%, 98%, 99% ormore sequence identity over a defined length of the molecules. As usedherein, “substantially homologous” also refers to sequences showingcomplete (100%) sequence identity to the polypeptide sequences.

In some embodiments, a sequence is not substantially homologous when itexhibits a sequence identity of 40% or less sequence identity.

“Sequence identity” as used herein refers to a relationship between twoor more polypeptide sequences, namely a reference polypeptide sequenceand a given polypeptide sequence to be compared with the referencepolypeptide sequence. Sequence identity is determined by comparing thegiven polypeptide sequence to the reference polypeptide sequence afterthe polypeptide sequences have been optimally aligned to produce thehighest degree of sequence similarity, as determined by the matchbetween strings of such sequences, with gaps introduced if necessary.Upon such alignment, sequence identity is ascertained on aposition-by-position basis, e.g., the sequences are “identical” at aparticular position if at that position, the amino acid residues areidentical. The total number of such position identities is then dividedby the total number of residues in the reference sequence to give %sequence identity.

Sequence identity can be readily calculated by known methods, includingbut not limited to, those described in Computational Molecular Biology,Lesk, A. N., ed., Oxford University Press, New York (1988),Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G.,Academic Press (1987); Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H.,and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988); the teachings ofwhich are incorporated herein by reference.

Methods to determine sequence identity are codified in publiclyavailable computer programs which determine sequence identity betweengiven sequences. Examples of such programs include, but are not limitedto, the GCG program package (Devereux, J., et al., Nucleic AcidsResearch, 12(1):387 (1984)), BLASTP, BLASTN and BLASTX (Altschul, S. F.et al., J. Molec. Biol., 215:403-410 (1990). The BLAST programs arepublicly available from NCBI and other sources (BLAST Manual, Altschul,S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J.Molec. Biol., 215:403-410 (1990), the teachings of which areincorporated herein by reference). These programs optimally alignsequences using default gap weights in order to produce the highestlevel of sequence identity between the given and reference sequences.

In specific embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide, such as at leasttwo, such as at least three, such as at least four, such as at leastfive, such as at least six, such as at least seven, such as at leastten, such as at least fifteen, such as at least twenty, such as at leasttwenty-five, such as at least twenty-six isolated polypeptidescomprising an amino acid sequence having a sequence identity of at least75%, such as at least 80%, such as at least 85%, such as at least 90%,such as at least 95%, such as at least 97%, such as at least 98%, suchas at least 99%, such as at least 100% to a polypeptide selected fromSEQ ID NOS: 1-27.

In other specific embodiments, the immunogenic composition of theinstant disclosure includes no more than one isolated polypeptide, suchas no more than two, such as no more than three, such as no more thanfour, such as no more than five, such as no more than six, such as nomore than seven, such as no more than ten, such as no more than fifteen,such as no more than twenty, such as no more than twenty-five, such asno more than twenty-six isolated polypeptides comprising an amino acidsequence having a sequence identity of at least 75%, such as at least80%, such as at least 85%, such as at least 90%, such as at least 95%,such as at least 97%, such as at least 98%, such as at least 99%, suchas at least 100% to an amino acid sequence selected from SEQ ID NOS:1-27.

In further specific embodiments, the immunogenic composition of theinstant disclosure includes at least one isolated polypeptide comprisingan amino acid sequence having a sequence identity of at least 75%, suchas at least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 1 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 2-27 or immunogenic fragments thereof.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 2 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1, 3 and 4-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 3 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1, 2 and 4-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 4 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-3 and 5-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 5 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-4 and 6-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 6 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-5 and 7-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 7 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-6 and 8-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 8 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-7 and 9-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 9 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-8 and 10-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 10 and optionally least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-9 and 11-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 11 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-10 and 12-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 12 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-11 and 13-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 13 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-12 and 14-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 14 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-13 and 15-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 15 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-14 and 16-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 16 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-15 and 17-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 17 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-16 and 18-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 18 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-17 and 19-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 19 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-18 and 20-27.

In some embodiments, the vaccine composition of the instant disclosureincludes at least one isolated polypeptide comprising an amino acidsequence having a sequence identity of at least 75%, such as at least80%, such as at least 85%, such as at least 90%, such as at least 95%,such as at least 97%, such as at least 98%, such as at least 99%, suchas at least 100% to SEQ ID NO: 20 and optionally at least one additionalisolated polypeptide, such as at least two, such as at least three, suchas at least four, such as at least five, such as at least six, such asat least seven, such as at least ten, such as at least fifteen, such asat least twenty, such as at least twenty-five, such as at leasttwenty-six additional isolated polypeptides comprising an amino acidsequence having a sequence identity of at least 75%, such as at least80%, such as at least 85%, such as at least 90%, such as at least 95%,such as at least 97%, such as at least 98%, such as at least 99%, suchas at least 100% to an amino acid sequence selected from SEQ ID NOS:1-19 and 21-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 21 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-20 and 22-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 22 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-21 and 23-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 23 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-22 and 24-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 24 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-23 and 25-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 25 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-24 and 26-27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 26 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-25 and SEQ ID NO: 27.

In some embodiments, the immunogenic composition of the instantdisclosure includes at least one isolated polypeptide comprising anamino acid sequence having a sequence identity of at least 75%, such asat least 80%, such as at least 85%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as at least 100% to SEQ ID NO: 27 and optionally at least oneadditional isolated polypeptide, such as at least two, such as at leastthree, such as at least four, such as at least five, such as at leastsix, such as at least seven, such as at least ten, such as at leastfifteen, such as at least twenty, such as at least twenty-five, such asat least twenty-six additional isolated polypeptides comprising an aminoacid sequence having a sequence identity of at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 97%, such as at least 98%, such as at least 99%,such as at least 100% to an amino acid sequence selected from SEQ IDNOS: 1-26.

In some embodiments, the polypeptides of the present disclosure arerecombinant polypeptides. The term “recombinant polypeptide” refers to aprotein produced by recombinant expression methods, such as, forexample, in prokaryotic or eukaryotic host cells, or in cell-free invitro expression systems.

The polypeptides of the present disclosure are typically expressed usingan expression vector and purified. Expression vectors may be eitherself-replicating extrachromosomal vectors or vectors that integrate intoa host genome. Generally, expression vectors include transcriptional andtranslational regulatory nucleic acid sequences, operably linked to thenucleic acid encoding the target protein.

In some embodiments, control sequences may be used for the expression ofan operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers. A nucleic acid sequence is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For example, DNA for a pre-sequence orsecretory leader is operably linked to DNA if it is expressed as apre-protein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Operably linked DNA sequences may be contiguousor non-contiguous. Methods for linking DNA sequences are well-known inthe art and include use of the polymerase chain reaction and ligation.The transcriptional and translational regulatory nucleic acid willgenerally be appropriate to the host cell used to express the targetprotein; for example, transcriptional and translational regulatorynucleic acid sequences from E. coli are typically used to express thetarget protein in E. coli.

Numerous types of appropriate expression vectors, and suitableregulatory sequences are known in the art for a variety of host cells.Methods for expressing polypeptides are well known in the art (e.g.,Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory; Berger and Kimmel (1987) Guideto Molecular Cloning Techniques, Methods in Enzymology, vol. 152,Academic Press, Inc., San Diego, Calif.; Ausubel et al. (1995) CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY) hereinincorporated by reference in their entireties.

The polypeptides of the present disclosure may be produced by culturinga host cell transformed with an expression vector containing nucleicacid encoding polypeptides of the present disclosure, such as encodingone or more of SEQ ID NOS: 1-27 or immunogenic fragments or variantsthereof, under the appropriate conditions to induce or cause expressionof the polypeptides of the present disclosure. The conditionsappropriate for protein expression will vary with the choice of theexpression vector and the host cell, and may be easily determined by oneskilled in the art using routine experimentation.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi,insect cells, and animal cells, including mammalian cells (such as humancells and cells lines). Thus, host cells include, but are not limitedto, Drosophila melanogaster cells, Tetrahymena, Saccharomyces cerevisiaeand other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293cells, Neurospora, BHK, CHO, COS, HeLa cells, Hep G2 cells, THP1 cellline (a macrophage cell line), and human embryonic kidney cell lines(e.g., HEK293).

Nucleic acid molecules encoding the polypeptides of the presentdisclosure may be cloned using standard molecular biological methods,including DNA amplification methods, such as the polymerase chain method(PCR) (see e.g., Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbour, N.Y.; Berger & Kimmel (1987)Methods in Enzymology. Vol. 152: Guide to Molecular Cloning Techniques,Academic Press, Inc., San Diego, Calif.; Co et al. (1992) J. Immunol.148:1149), which are each herein incorporated by reference. Thus, forexample, a nucleic acid molecule encoding a polypeptide of the presentdisclosure may be PCR-amplified using a sense primer containing onerestriction site and an antisense primer containing another restrictionsite. This will produce a nucleic acid encoding the desired sequence orsubsequence having terminal restriction sites. This nucleic acid canthen readily be ligated into a vector having appropriate correspondingrestriction sites. Suitable PCR primers may be chosen by one of skill inthe art based on the sequence to be expressed. Appropriate restrictionsites can also be added by site-directed mutagenesis as is well known inthe art.

The methods of introducing exogenous nucleic acids into host cells arealso well known in the art, and will vary with the host cell used.Suitable techniques include, but are not limited to, dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, viral infection,encapsulation of the nucleic acids in liposomes, and directmicroinjection of nucleic acids into nuclei.

The polypeptides of the present disclosure may be isolated or purifiedin a variety of ways known to those skilled in the art depending on whatother components are present in the sample. Standard purificationmethods include electrophoretic, molecular, immunological, andchromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography.

Some embodiments of the disclosure provide synthetic polypeptides of thepresent disclosure. Polypeptides having up to about 100-150 amino acidresidues may be prepared by in vitro synthesis using establishedtechniques. Synthetic polypeptides may be prepared by chemical synthesis(such as solid phase peptide synthesis) using methods known in the art.

In some embodiments, the immunogenic composition of the presentdisclosure includes a pharmaceutically acceptable carrier. The carriermust be “acceptable” in the sense that it is compatible with the activeingredient of the composition, and typically, capable of stabilizing theactive ingredient and not deleterious to the subject to be treated. Insome embodiments, the pharmaceutically acceptable carrier is anon-naturally occurring pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers (vehicles) may be conventional,but are not limited to conventional carriers (vehicle). For example, E.W. Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa., 15th Edition (1975) and D. B. Troy, ed. Remington: TheScience and Practice of Pharmacy, Lippincott Williams & Wilkins,Baltimore Md. and Philadelphia, Pa., 21st Edition (2006) describecompositions and formulations suitable for pharmaceutical delivery ofone or more molecules and additional pharmaceutical agents.

The present immunogenic compositions may comprise buffers (e.g., sodiumphosphate, histidine, potassium phosphate, sodium citrate, potassiumcitrate, maleic acid, ammonium acetate,tris-(hydroxymethyl)-aminomethane (tris), acetate, diethanolamine,etc.), amino acids (e.g., argenine, cysteine, histidine, glycine,serine, lysine, alanine, glutamic acid, proline), sodium chloride,potassium chloride, sodium citrate, sucrose, glucose, mannitol, lactose,glycerol, xylitol, sorbitol, maltose, inositol, trehalose, bovine serumalbumin (BSA), albumin (e.g., human serum albumin, recombinant albumin),dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine,gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC),polyethylene glycol (PEG), ethylene glycol, dimethylsulfoxide (DMSO),dimethylformamide (DMF), hydrochloride, sacrosine, gamma-aminobutyricacid, Tween-20, Tween-80, sodium dodecyl sulfate (SDS), polysorbate,polyoxyethylene copolymer, sodium acetate, ammonium sulfate, magnesiumsulfate, sodium sulfate, trimethylamine N-oxide, betaine, zinc ions,copper ions, calcium ions, manganese ions, magnesium ions, CHAPS,sucrose monolaurate, 2-O-beta-mannoglycerate, the like, or a combinationthereof.

In some embodiments, the present immunogenic compositions may comprisepropellants (e.g., hydrofluoroalkane (HFA)) for aerosol delivery. Insome embodiments, the immunogenic compositions of the present disclosuremay be formulated as described in U.S. Pat. No. 5,192,743 that form agel when reconstituted and which can improve stability of a protein ofinterest (e.g., for storage).

Immunogenic compositions of the present disclosure may be appropriatelyconstructed for some or all routes of administration, for exampletopical administration (including inhalation and nasal administration),oral or enteral administration, intravenous or parenteraladministration, transdermal administration, epidural administration orthe like. For example, parenteral formulations usually compriseinjectable fluids that include pharmaceutically and physiologicallyacceptable fluids such as water, physiological saline, balanced saltsolutions, aqueous dextrose, glycerol or the like as a vehicle. Forsolid compositions (for example, powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, immunogeniccompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

In some embodiments, a parenteral formulation may comprise injectablefluids that include pharmaceutically and physiologically acceptablefluids such as water, physiological saline, balanced salt solutions,aqueous dextrose, glycerol or the like as a vehicle.

For solid compositions (for example, powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

The aforementioned immunogenic compositions and protein modifications toincrease protein stability can be applied as described in U.S. PatentApplication 2009/032692, which is herein incorporated by reference inits entirety.

In some embodiments the pharmaceutically acceptable carrier of thepresent disclosure includes an adjuvant, such as a non-naturallyoccurring adjuvant. As used herein, an “adjuvant” is understood as apharmacological or immunological agent that modifies the effect of otheragents (e.g., immunogen or target antigen in an immunogenic composition)while having few if any direct effects when given by itself. In someembodiments, an adjuvant will enhance the recipient's immune response tothe polypeptides in the present immunogenic composition while keepingthe injected foreign material at a minimum.

Suitable adjuvants are well known in the art (see, for example, VaccineDesign-The Subunit and Adjuvant Approach (1995) PharmaceuticalBiotechnology, Volume 6 (eds. Powell, M. F., & Newman, M. J.) PlenumPress, New York and London, ISBN 0-306-44867-X), which is incorporatedherein by reference in its entirety. Examples of adjuvants include, butare not limited to, alum-precipitate, Freund's complete adjuvant,Freund's incomplete adjuvant, monophosphoryl-lipid A/trehalosedicorynomycolate adjuvant, water in oil emulsion containingCorynebacterium parvum and tRNA, and other substances that accomplishthe task of increasing immune response by mimicking specific sets ofevolutionarily conserved molecules including liposomes,lipopolysaccharide (LPS), molecular cages for antigen, components ofbacterial cell walls, and endocytosed nucleic acids such asdouble-stranded RNA, single-stranded DNA, and unmethylated CpGdinucleotide-containing DNA. Other examples include cholera toxin, E.coli heat-labile enterotoxin, liposome, immune-stimulating complex(ISCOM), immunostimulatory sequences oligodeoxynucleotide, and aluminumhydroxide.

Other exemplary adjuvants include the adjuvants described in Lanar etal., U.S. Pat. No. 7,029,685 and U.S. Patent Publication No.2006/0073171, herein incorporated by reference in their entireties.Alternatively, the polypeptides of the immunogenic composition describedherein can be used without any adjuvant.

Methods

The present disclosure is further directed to methods for preventing ortreating filarial diseases. As used herein, “filarial diseases” refer todiseases caused by thread-like nematodes (filariae) that belong to theroundworm superfamily filarioidea family Filariidae. Such diseasesinclude, but are not limited to lymphatic filariasis, river blindness,loiasis or heartworm. As used herein “preventing” refers to theadministration of a therapeutically effective amount of a polypeptide,immunogenic composition, such as a vaccine, of the present disclosure toan animal in order to protect the animal from the development of, forexample, lymphatic filariasis, river blindness, loiasis, heartworm orthe symptoms thereof. In some embodiments, the immunogenic compositionof the disclosure is administered to a subject that is at risk fordeveloping a lymphatic filariasis, river blindness, loiasis orheartworm.

By “treating” a disease associated with filarial infection such aslymphatic filariasis, river blindness, loiasis or heartworm is intendedadministration of a therapeutically effective amount of a polypeptide,immunogenic composition, such as a vaccine of the present disclosure toan animal that has, for example, lymphatic filariasis, river blindness,loiasis or heartworm or that has been exposed to a filarial infection,such as a filarial worm selected from Brugia malyai, Wuchereriabancrofti, Onchocerca volvulus, Loa loa, or Dirofilaria immitis, wherethe purpose is to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the condition or the symptoms of thelymphatic filariasis, river blindness, loiasis or heartworm.

The immunogenic compositions of the present disclosure may beadministered to a subject. In some embodiments, the subject is a mammal,e.g., a non-human primate such as a baboon or macaque. In someembodiments, the subject mammal is a human. The subject mammal can alsoinclude, but is not limited to, pet or companion animals (e.g. cats,dogs, house rabbits, ferrets, rodents, including gerbils, hamsters,chinchillas, rats, mice, guinea pigs, etc); working animals, such asguide animals (e.g., monkeys; herding animals, etc.), draught animals(e.g., draught horses, oxen, camels, elephants, oxen, camels, donkeys)and sport animals (e.g., racing or show-jumping horses); livestock(e.g., alpaca, banteng, bison, camel, cattle, deer, donkey, gayal, goat,llama, mule, pig, reindeer, sheep, water buffalo, yak, etc.); laboratoryanimals (e.g., mice, rabbits, rats, non-human primates); andundomesticated animals held in captivity, e.g. in zoological parks andthe like. In other embodiments, particularly when the disease beingprevented or treated is heartworm, the subject is a dog or a cat,typically a dog.

In various embodiments, a therapeutically effective amount of theimmunogenic composition described herein is administered to the subject.As used herein “a therapeutically effective amount” is an amount thatprovides a therapeutic effect for a given condition and administrationregimen. In particular aspects of the disclosure, a “therapeuticallyeffective amount” refers to an amount of a polypeptide, immunogeniccomposition, or vaccine of the present disclosure that when administeredto an animal brings about a positive therapeutic response with respectto the prevention or treatment of a subject for diseases or conditionsassociated with filarial worms such as Brugia malyai, Wuchereriabancrofti, Onchocerca volvulus, Loa loa, or Dirofilaria immitis. Forexample, a positive therapeutic response in regard to treating diseasesor conditions associated with filarial worms includes curing orameliorating the symptoms of the disease.

A positive therapeutic response with respect to preventing a conditionassociated with a filarial infection includes, for example, theproduction of filarial antibodies by the subject in a quantitysufficient to protect against development of the disease. The productionof antibodies elicited by a treatment is readily ascertained byobtaining a plasma or serum sample from the subject to which animmunogenic composition is administered, and assaying the antibodiestherein for their ability to bind to the polypeptide(s) used to elicitthe immune response to filarial worms. Exemplary methods include, butare not limited to, ELISA assays, immunofluorescence assays (IFA), orother immunoassays such as a Western blots, as is well known in the art.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors that may be taken into account include theprevalence of filarial worms in the geographical vicinity of thepatient, the severity of the disease state of the patient, age, andweight of the patient, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. An appropriate effective amount may be readily determined usingonly routine experimentation. Several doses may be needed per individualin order to achieve a sufficient response to effect treatment. Suitableregimes for initial administration and follow-up administration (e.g.,booster shots) are also variable, but are typified by an initialadministration followed in intervals (weeks or months) by a subsequentadministration. In some embodiments, typical dosages may range fromabout 0.01 to about 20 mg/kg, and more particularly from about 0.1 toabout 10 mg/kg.

EXAMPLES Example 1. Materials and Methods

Dissections

Adult Brugia malayi worms were received in multiple shipments from TRSLabs (Athens, Ga.) and frozen at −80° C. until processing. Forseparation of anatomic structures, worms were thawed at room temperatureand then dissected using a stereomicroscope and fine tipped forceps. Oneset of forceps was used to grip and steady the center of the parasiteafter thawing and placement into a petri dish filled with phosphatebuffered saline (PBS). Another set of forceps was used to grasp andgently twist the parasite close to the first set of forceps, resultingin a tear of the body wall. The cephalic tip of the body wall was thengrasped and gently peeled away from the rest of the organs. The caudalportion of the body wall was then peeled away from the intestines anduterine tubes (FIG. 2). Reproductive organs were identified by theiranterior junction and then separated from the intestine. Each anatomicfraction (intestine, reproductive tract, and body wall) was placed in amicrocentrifuge tube filled with PBS. These were stored at −20° C. untilprotein extraction.

Protein Extraction

The samples were thawed and then centrifuged in 1.5 ml eppendorf tubes.The pelleted tissues were frozen and thawed 4 times by cycling throughplacement on dry ice for 10 minutes followed by placement in a 37° C.water bath. Using a mini disposable micropestle, the samples werehomogenized with 50 μl of UPX extraction buffer (Expedeon). Themicropestle was washed with 50 μl of UPX extraction buffer and processedas per the manufacturer's instructions. In brief, samples were placed ina 100° C. water bath for 5 minutes, removed and cooled at 4° C. for onehour. Samples were then centrifuged at 15,000×g for 10 minutes andsupernatant was collected.

Protein Concentrations were measured by BCA assay. 400 μg proteins ofintestine, body wall and reproductive tract each were reduced, alkylatedand trypsin digested overnight following filter-aided digestionprocedure using a FASP digestion kit (Protein Discovery, San Diego,Calif.) according to vendor protocol. Tryptic peptides were furtherdesalted, lyophilized and reconstituted in 25% acetonitrile with 0.1%formic acid and further fractionated using strong cation exchange (SCX)chromatography. The SCX fractions of the three samples were pooled into16 to 18 fractions each, lyophilized and reconstituted in 0.1%trifluoroacetic acid to be analyzed by liquid chromatography-massspectrometry (LC-MS).

Nanobore Reversed-Phase Liquid Chromatography Tandem MS (nanoRPLC-MSMS)

Nanobore RPLC-MSMS was performed using an Agilent 1200 nanoflow LCsystem coupled online with a LTQ Orbitrap XL mass spectrometer. The RPLCcolumn (75 μm i.d.×10 cm) were slurry-packed in-house with 5 μm, 300 Åpore size C-18 stationary phase into fused silica capillaries with aflame pulled tip. After sample injection, the column was washed for 20minutes with 98% mobile phase A (0.1% formic acid in water) at 0.5μl/min. Peptides were eluted using a linear gradient of 2% mobile phaseB (0.1% formic acid in acetonitrile) to 35% B in 100 minutes, then to80% B over an additional 40 minutes. The column flow-rate was maintainedat 0.25 μl/min throughout the separation gradient. The mass spectrometerwas operated in a data-dependent mode in which each full MS scan wasfollowed by seven MS/MS scans wherein the seven most abundant molecularions were dynamically selected for collision-induced dissociation (CID)using a normalized collision energy of 35%.

Protein Identification

The LC-MS/MS data were searched using SEQUEST through Bioworks interfaceagainst a combined database of Brugia malayi database downloaded fromThe Institute for Genomic Research (TIGR) and the Wolbachia databasefrom New England Biolabs (Beverly, Mass.). SEQUEST was searched with afragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 25PPM. Carbamidomethyl of cysteine was specified in SEQUEST as a fixedmodification. Oxidation of methionine was specified in SEQUEST as avariable modification. Scaffold (version Scaffold 3.5.2, ProteomeSoftware Inc., Portland, Oreg.) was used to validate MS/MS based peptideand protein identifications. Peptide identifications were accepted ifthey could be established at greater than 95.0% probability by thePeptide Prophet algorithm (Keller et al., 2002, Anal. Chem., 74,5383-5392, herein incorporated by reference). Protein identificationswere accepted if they could be established at greater than 99.0%probability and contained at least 2 identified peptides. Proteinprobabilities were assigned by the Protein Prophet algorithm(Nesvizhskii et al., 2003, Anal. Chem., 75, 4646-4658, hereinincorporated by reference). Proteins that contained similar peptides andcould not be differentiated based on MS/MS analysis alone were groupedto satisfy the principles of parsimony. TIGR accession numbers werematched to PUB_loci from the proteome published by Bennuru et. Al.,2011, Proc. Natl. Acad. Sci. U.S.A., 108, 9649-9654, herein incorporatedby reference.

Heat Map Analysis

A heat map of the relative abundance of each protein, defined as thenumber of unique matching peptides within each anatomic fraction, wasmade with JMP software.

Quantitative Analysis

Protein quantitation was determined by normalized spectral abundance.This approach provides a theoretical quantitative value useful fordetermining relative abundance of a single protein between samples(McIlwain et al., 2012, BMC Bioinformatics, 13, 308, Paoletti et al.,2006, Proc. Natl. Acad. Sci. U.S.A., 103, 18928-18933) and an estimationof relative abundance between different proteins in one sample (Liu etal., 2004, Anal. Chem., 76, 4193-4201). Exclusive spectral counts,spectra that match to only 1 protein, were first divided by the lengthof the protein to account for the differences in numbers of possiblespectra. This calculation provides the spectral abundance factor. Thiswas then normalized to obtain the normalized spectral abundance factor(NSAF) by dividing by the sum of the total spectral abundance factorsfound within that anatomic fraction.

${NSAF} = {\frac{( \frac{Spectra}{Length} )p}{\sum\limits_{p = 1}^{n}{( \frac{Spectra}{length} )p}}.}$NSAF enrichment was then calculated by dividing the NSAF of a givenprotein in the target fraction divided by the sum of the NSAF of theother two fractions to determine whether a protein was more abundant or“enriched” in one fraction compared to the others.

${{NSAF}\mspace{14mu}{enrichment}} = {\frac{{NSAF}\mspace{14mu}( {{target}\mspace{14mu}{fraction}} )}{{NSAF}\mspace{14mu}{of}\mspace{14mu}{other}\mspace{14mu}{two}\mspace{14mu}{fractions}\mspace{14mu}({added})}.}$Proteins were considered enriched when they had an NSAF enrichment valueof 2 or greater.

Functional Categories for Gene Set Enrichment Analysis (GSEA)

The proteome of B. malayi had previously been functionally characterizedby Bennuru and colleagues (Bennuru, et al., 2011). For proteinspreviously annotated for function, no further analysis of function wascarried out. The 665 newly identified proteins were annotated basedloosely on the KOG and PFAM functions. Categories of function were usedas previously described (Bennuru, et al., 2011), including cytoskeletal,extracellular matrix, immunological, metabolism, nuclear regulation,protein export, protein modification, protein synthesis, signaltransduction, transcription, transporters, and uncharacterized.Functions of anatomic fractions were analyzed based on GSEA, whichanalyzes the data for bias in a condition (or anatomic fraction)(Subramanian et al., 2005, Proc. Natl. Acad. Sci. U.S.A., 102,15545-15550, herein incorporated by reference). Proteins were rankedaccording to abundance using spectral counts. A priori defined sets ofproteins, based on functional annotation, were then analyzed using GSEAfor bias within each anatomic fraction.

BLASTp

BLASTp was performed on proteins of interest from B. malayi to identifysimilarity among W. bancrofti, O. volvulus, D. immitis, L. boa, and H.sapiens. BLAST query was conducted with blast+ 2.2.29 downloaded fromNCBI. Protein databases for W. bancrofti, O. volvulus, H. sapiens, L.loa were downloaded from uniprotKB. Protein database for D. immitis wasdownloaded from nematodes.org. A FASTA file containing the B. malayiproteins of interest were blasted against each other genomeindividually. Percent identity and query coverage were recorded for thetop scoring sequence for each protein. Score is determined by analgorithm that takes into account similarity of AA sequence, gaps inhomologous regions, and length of homology. Percent identity is definedas the percentage of amino acids that match perfectly over the sequenceregion with greatest homology.

Example 2. Results

Distinct Anatomic Fractions Exhibit Markedly Different Expression ofProteins

Based on a match of 2 unique peptides to a protein, we identified atotal of 5023 proteins. Of these, 204 were Wolbachia proteins, and 34could be matched to more than 1 specific protein, leaving 4,785specifically identified B. malayi proteins. While 1,895 of the proteinswere identified by two peptides in all three anatomical fractions of theparasite, 396 proteins were identified solely within the intestine, 114solely within the body wall, and 1011 solely within the uterine tubes(data not shown). Additionally, although the majority of proteins werepresent in all three anatomic fractions, we found that there was adifferential expression of each protein among the anatomic fractions(FIG. 3).

Proteomic Profiling of the B. malayi Intestine is Consistent withFunctional Absorption and Digestion

Like all nematodes, filarial parasites have a fully formed intestine.However, the functionality of this tract is not completely clear (Munn,E. A. and Munn, P. D., 2002, Feeding and Digestion. In: Lee, D. L.,(Ed., The Biology of Nematodes. CRC Press Taylor and Francis Group, BocaRaton, Fla., pp. 211-233, herein incorporated by reference). Weperformed several analyses to further elucidate the possible function ofthe intestine in B. malayi. First, gene set enrichment analysis (GSEA)was performed which showed a bias for proteins with transporter functionto be present within the intestine (FIG. 4). Next, we rank ordered theproteins that were enriched within the intestine based on their NSAFvalue, a measure which takes into account the number of spectra uniquelymatching to a protein and the length of the protein in amino acids.Spectral counting has previously been shown to be useful to determinerelative abundance of a single protein in different samples (McIlwain,et al., 2012, Paoletti, et al., 2006) and provide a reasonableapproximation of protein abundance within a sample compared to otherproteins in the same sample (Liu, et al., 2004). Of the 20 mostabundant, enriched, and named intestine proteins, 3 are proteolyticenzymes (Bm1_00205, Bm1_18805, Bm1_34740), 2 are transporters(Bm1_42930, and Bm1_24840), and 1 is associated with phagocytosis(Bm1_02265). The abundance of such proteins suggests the intestine isinvolved in both digestion and active absorption of nutrients. Of theremaining 20 most abundant named proteins in the intestine, 3 are muscleassociated proteins (Bm1_28910, Bm1_45035, Bm1_00655) and the rest areinvolved in various functions including translation, cell trafficking,RNA binding, cell adhesion, hydrolysis, lipid metabolism, catabolism,and cellular structure.

Table 1, below, depicts the twenty most abundant proteins, with propernames, enriched in the intestine of adult female B. malayi based onnormalized spectral abundance factor (NSAF). The NSAF enrichment valuesare also depicted. “Specific” means that the protein was only foundwithin the intestine.

TABLE 1 Abundance NSAF NSAF enrichment Protein Type Accession NameIntestine Intestine Translational Bm1_41515 40S ribosomal protein S21,putative 5.4E−03 2.17 Muscle Bm1_28910 Calsequestrin, skeletal muscle2.5E−03 2.90 Associated isoform precursor, putative Cell traffickingBm1_14235 SNARE domain containing protein 2.4E−03 2.0 Muscle Bm1_45035Probable myosin regulatory light 2.2E−03 2.5 Associated chain, putativeProtease Bm1_34740 aspartic protease BmAsp-1, 1.1E−03 16.0 identicalcarrier protein Bm1_21135 Acyl CoA binding protein 9.0E−04 7.68 MuscleBm1_00655 myosin heavy chain, nonmuscle 7.8E−04 2.0 Associated type 1,putative Phagocytosis Bm1_02265 MGC69076 protein-related 7.3E−04 3.77associated Xenobiotic Bm1_13480 UDP-glucoronosyl and UDP- 7.0E−04 28.16metabolism glucosyl transferase family protein RNA binding Bm1_20295Glycine-rich RNA-binding protein.- 6.9E−04 8.96 related MiscellaneousBm1_25280 Prion-like--related 6.4E−04 2.37 Cell Adhesion Bm1_10500 AMOPdomain containing protein 6.1E−04 5.99 Hydrolase Bm1_24820 Histidineacid phosphatase family 6.1E−04 6.32 protein Cytoskeleton Bm1_30265Tubulin alpha chain, putative 5.9E−04 2.95 Transporter Bm1_42930Excitatory amino acid transporter, 5.7E−04 2.75 putative Lipid Bm1_08150NAD-dependent malic enzyme, 5.5E−04 7.10 Metabolism mitochondrialprecursor, putative Catabolism Bm1_48185 putative amidase 5.1E−04 3.74Transporter Bm1_24840 Major Facilitator Superfamily 4.7E−04 19.88protein Protease Bm1_18805 Papain family cysteine protease 4.4E−04Specific containing protein Protease Bm1_00205 ShTK domain containingprotein 4.2E−04 3.42

Many Predominant Body Wall Enriched Proteins Provide Muscular Structureor are Involved in Muscular Contraction

The body wall of B. malayi includes, from superficial to deep, theepicuticle, cuticle, epidermis, musculature (divided into a superficialfibrous portion and a deeper metabolically active portion) and thelateral cords (FIG. 4). The musculature is separated into quadrants bythe lateral, ventral and dorsal cords with up to 9 myocytes per quadrant(Vincent et al., 1975, J. Parasitol., 61, 499-512.). The lateral cordscontain the cell bodies of the epidermis, which produces and maintainsthe cuticle. Also associated with the lateral cords is a secretory glandwhich is connected to the secretory pore by the secretory canal(Landmann et al., 2010, PLoS Negl. Trop. Dis., 4, e758) hereinincorporated by reference. The ventral and dorsal cords are associatedwith nerves that innervate the musculature.

GSEA of the body wall showed a bias for cytoskeletal proteins andproteins of immunological interest to be present within the body wall(FIG. 5). Further, analysis of the 20 most abundant named proteins thatwere enriched within the body wall by NSAF yielded 12 proteinsassociated with muscle structure or regulation of muscular contraction(Table 2). These included an actin (Bm1_21705), 4 myosins (Bm1_40715,Bm1_50805, Bm1_00935, Bm1_14060), 2 paramyosins (Bm1_04450, Bm1 02615),1 tropomyosin (Bm1_02060), and a disorganized muscle protein(Bm1_40320). In addition to the muscular proteins, there were 3cuticular proteins, a glutathione peroxidase, which provides protectionfrom oxidative damage, a cytoskeletal protein, a heat shock protein, anda glutamine synthetase.

Table 2, below, depicts the twenty most abundant proteins enriched inthe body wall of adult female Brugia malayi.

TABLE 2 Abundance NSAF (NSAF) enrichment Protein type Accession NameBody Wall Body Wall Muscle Bm1_21705 actin 1, putative 6.2E−02 3.86associated Cytoskeletal Bm1_45215 intermediate filament protein,putative 2.0E−02 2.94 Muscle Bm1_40320 Disorganized muscle protein 1,1.6E−02 5.15 associated putative HSP Bm1_19805 small heat shock protein,putative 1.4E−02 7.97 Muscle Bm1_04450 Paramyosin, putative 1.1E−02 5.87associated Muscle Bm1_02615 Paramyosin, identical 1.0E−02 6.33associated Calcium Bm1_48810 EF hand family protein 6.5E−03 9.81 BindingCuticle Bm1_13015 Nematode cuticle collagen N-terminal 6.1E−03 3.05domain containing protein Muscle Bm1_01235 Tropomyosin-related 6.0E−035.24 associated Muscle Bm1_49075 Calponin homolog OV9M, putative 5.9E−033.31 Associated Muscle Bm1_40715 myosin heavy chain, putative 5.8E−033.11 associated Cuticle Bm1_54705 Nematode cuticle collagen N-terminal5.5E−03 6.57 domain containing protein Muscle Bm1_50805 Myosin tailfamily protein 4.7E−03 4.00 associated Antioxidant Bm1_40465 Cuticularglutathione peroxidase 4.5E−03 2.59 precursor, putative Muscle Bm1_00935myosin heavy chain B (MHC B), 4.4E−03 3.36 associated putativeCarbohydrate Bm1_16060 carbohydrate phosphorylase, putative 4.2E−03 2.67metabolism Muscle Bm1_14060 myosin heavy chain B (MHC B), 4.1E−03 2.23associated putative Cuticle Bm1_17485 Nematode cuticle collagenN-terminal 3.2E−03 2.40 domain containing protein Muscle Bm1_02060Tropomyosin family protein 3.2E−03 3.19 associated Amino Acid Bm1_53470glutamine synthetase, putative 3.0E−03 3.16 Synthesis

Nuclear Regulatory Proteins, Including Those Involved in ChromatinOrganization are Enriched and Highly Abundant in the Reproductive Tract

The nematode female reproductive tract consists of two ovaries wheregamete production takes place, two seminal receptacles (aka spermatheca)which store sperm obtained from males, and 2 uterine tubes that allowfor embryo and subsequent in utero microfilaria development (FIG. 1).The two uterine tubes merge into the vulva, which is on the ventralsurface of the worm in the cephalic region (Fischer et al., 2011, PLoSNegl. Trop. Dis., 5, e1174. Jiang et al., 2012, Int. J. Parasitol., 42,841-850, Landmann, et al., 2010, Li et al., 2012, BMC Genomics, 13, 184,which are herein incorporated by reference in their entireties). GSEAshowed a bias for transcription and nuclear regulation proteins to bepresent within the female reproductive tract (FIG. 6). Similarly the 20most abundant named proteins that were enriched in the reproductivetract as assessed by NSAF contained many proteins involved in nuclearregulation. 12 proteins contained domains associated with nucleotidebinding or splicing, with 8 of these 12 being histones or histonelinkers (Bm1_02505, Bm1_02515, Bm1_20280, Bm1_02495, Bm1_, 20285, Bm1_,38685, Bm1_02800, Bm1_04110). Three microfilarial sheath proteins werealso abundant and enriched within the reproductive tract, which isconsistent with presence of developing microfilariae within the uterinetubes. The remaining 7 proteins are involved in trafficking, protectionfrom oxidation, xenobiotic metabolism, proteolysis and cell adhesion.

Table 3, below, depicts the most abundant enriched named proteins in thereproductive tract of adult female Brugia malayi. Specific means thatthe protein was only found within the reproductive tract.

TABLE 3 Abundance NSAF NSAF Enrichment Reproductive Reproductive ProteinType tract tract Chromatin Bm1_02505 histone H2A, putative 3.4E−02 8.1organization Chromatin Bm1_02515 histone H4, putative 3.1E−02 2.7organization Chromatin Bm1_20280 Probable histone H2B 3, 1.1E−02 4.3organization putative Chromatin Bm1_02495 histone H3, putative 7.8E−0325.9 organization Sheath Bm1_19100 Major microfilarial sheath 6.2E−032.6 protein precursor.-related Chromatin Bm1_20285 histone H2A, putative5.9E−03 17.2 organization Chromatin Bm1_38685 Histone H2A variant,putative 3.4E−03 2.7 organization Sheath Bm1_05185 sheath protein 5,identical 2.8E−03 2.9 Trafficking Bm1_07925 peroxisomal membrane 2.2E−032.6 anchor protein, putative Antioxidant Bm1_44840 GlutathioneS-transferase, N- 2.0E−03 2.3 terminal domain containing protein DNABm1_25620 high mobility group protein, 1.7E−03 10.2 binding putativeSheath Bm1_00650 microfilarial sheath protein, 1.2E−03 2.4 identical RNABm1_49560 NOP5/NOP58, putative 1.2E−03 2.2 splicing RNA Bm1_49460 smallnuclear 1.1E−03 2.3 modulation ribonucleoprotein-associated proteinhomolog F9F13.90 - Arabidopsis thaliana, putative Chromatin Bm1_57630retinoblastoma-binding 1.1E−03 2.6 organization protein., putativeChromatin Bm1_04110 linker histone H1 and H5 9.6E−04 4.7 organizationfamily protein Xenobiotic Bm1_32235 Flavin-binding 9.4E−04 2.6metabolism monooxygenase-like family protein Chromatin Bm1_02800 HistoneH2B 2, putative 9.0E−04 Specific organization Protease Bm1_45620 Trypsinfamily protein 8.9E−04 36.5 Cell Bm1_17270 Fasciclin domain containing8.1E−04 2.8 Adhesion protein

Identification of Potential Intestine Vaccine Candidates

To identify gastrointestinal proteins that could potentially be used asvaccine candidates, we analyzed the proteomics set for proteins thatwere enriched in the intestine, had at least one predicted transmembranedomain, and were not predicted to be in the mitochondria. We soughtluminal surface proteins because these proteins may be accessible tohost antibodies after vaccination. 106 proteins were identified withthese criteria (Table A and Table B, below, provided after the Examples)and were categorized based on likely metabolic function within the cell.The amino acid sequences identified by Genbank Accession numbers inTable. A and Table B are herein incorporated by reference.

In order to evaluate the potential of the 106 surface proteins aspan-filarial vaccine candidates, we performed a blast search for eachprotein against databases for Homo sapiens, Wuchereria bancrofti, whichcauses lymphatic filariasis, Onchocerca volvulus, which causes riverblindness, L. loa, which causes loiasis, and Dirofilaria immitis, whichcauses heartworm. Proteins were selected that contained a percentidentity of >75% to W. bancrofti or O. volvulus and <40% homology tohumans. 72 proteins matched these criteria.

We then selected those proteins that had 1-2 transmembrane domains forease of recombinant protein production. These were evaluated withInterpro software for the presence of non-cytoplasmic domains that couldbe bound by host antibodies. 27 proteins matched all of these criteria(Tables C and D, below, provided after the Examples), with 12 displayingsubstantial homology between all of the filarial species. Of these 27proteins, 10 are hypothetical proteins, 3-4 are proteases, 2 areinvolved in xenobiotic metabolism using glucuronidation, 2 participatein cell adhesion, 2 function in cell signaling, and 2 are chaperones.

Certain Excretory/Secretory (ES) Products are Associated with SpecificAnatomic Fractions

227 proteins were found within the ES product of adult female B. malayiin a previous study by Bennuru and colleagues (Bennuru et al., 2009,PLoS Negl. Trop. Dis., 3, e410, herein incorporated by reference). Tobetter define the origin of these proteins, we analyzed all adult femaleES proteins for enrichment within any of the three worm fractions fromthis study. Four (1.7%) of these proteins were either enriched orspecific to the intestine (Table 4). The most notable of these was thepapain family cysteine protease (Bm1_18805). Eight (3.5%) female ESproducts were enriched within the body wall (Table 5), including twoproteins that protect against oxidative damage, cuticular glutathioneperoxidase (Bm1_40465) and peptide methionine sulfoxide reductase(Bm1_10795) (Weissbach, et al., 2005). Other ES products enriched withinthe body wall included a cuticle collagen (Bm_13015), and muscularproteins.

There were 30 adult female ES products (13%) enriched within the femalereproductive tract (Table 6). Some of these antigens include Juv-p120(Bm1_18010), which has been implicated in being critical for MFsurvival, Von willebrand factor type A domain containing protein(Bm1_27495), which likely binds to collagen, a trypsin inhibitor(Bm1_03520), and an aspartyl amino peptidase (Bm1_16690).

Tables 4, 5, and 6 below depict the intestine-enriched, body-wallenriched, and reproductive tract-enriched ES products, respectively.“Specific” means that the protein was only was only identified withinthe specified anatomical fraction.

TABLE 4 Abundance NSAF (NSAF) Enrichment ID Name Body wall Body wallBm1_13015 Nematode cuticle collagen 6.1E−03 3.05 N-terminal domaincontaining protein Bm1_50805 Myosin tail family protein 4.7E−03 4.00Bm1_40465 Cuticular glutathione 4.5E−03 2.59 peroxidase precursor,putative Bm1_39425 protein unc-22, putative 8.8E−04 7.42 Bm1_26690Prion-like-, putative 8.0E−04 4.73 Bm1_12515 Immunoglobulin I-set4.5E−04 7.68 domain containing protein Bm1_45145 Ryanodine Receptor1.4E−04 3.67 TM 4-6 family protein Bm1_10795 Peptide methionine 9.6E−05Specific sulfoxide reductase family protein

TABLE 5 Abundance NSAF (NSAF) Enrichment ID Name Body wall Body wallBm1_13015 Nematode cuticle collagen N- 6.1E−03 3.05 terminal domaincontaining protein Bm1_50805 Myosin tail family protein 4.7E−03 4.00Bm1_40465 Cuticular glutathione 4.5E−03 2.59 peroxidase precursor,putative Bm1_39425 protein unc-22, putative 8.8E−04 7.42 Bm1_26690Prion-like-, putative 8.0E−04 4.73 Bm1_12515 Immunoglobulin I-set domain4.5E−04 7.68 containing protein Bm1_45145 Ryanodine Receptor 1.4E−043.67 TM 4-6 family protein Bm1_10795 Peptide methionine sulfoxide9.6E−05 Specific reductase family protein

TABLE 6 Abundance NSAF (NSAF) Enrichment Reproductive Reproductive IDName Tract Tract Bm1_29260 60S ribosomal protein L34, putative 4.6E−042.4 Bm1_20440 Biotin/lipoate A/B protein ligase family 4.5E−04 2.8protein Bm1_43080 hypothetical protein 3.7E−04 2.1 Bm1_02485 Potentialglobal transcription activator 2.5E−04 2.6 SNF2L, putative Bm1_16970hypothetical protein 1.8E−04 2.7 Bm1_16690 Aspartyl aminopeptidase,putative 1.6E−04 3.4 Bm1_18010 excretory/secretory protein Juv-p1201.4E−04 3.4 precursor-related Bm1_21390 RNA binding protein, putative1.3E−04 2.0 Bm1_48000 RAS FAMILY PROTEIN 1.1E−04 2.7 Bm1_12225 G-patchdomain containing protein 1.0E−04 3.9 Bm1_03520 Kunitz/Bovine pancreatictrypsin inhibitor 9.9E−05 2.8 domain containing protein Bm1_02770Galactosyltransferase family protein 8.5E−05 2.6 Bm1_48025Alpha-catulin, putative 7.5E−05 3.5 Bm1_41495 Gex interacting proteinprotein 4, isoform c- 7.2E−05 7.0 related Bm1_18480 HYPOTHETICAL PROTEIN7.2E−05 Specific Bm1_46460 26S PROTEASOME REGULATORY 6.4E−05 5.4 CHAIN4, PUTATIVE Bm1_49790 tRNA modification GTPase TrmE family 5.1E−05 2.0protein Bm1_27495 von Willebrand factor type A domain 4.9E−05 2.1containing protein Bm1_46930 HYPOTHETICAL PROTEIN 4.4E−05 2.1 Bm1_21025hypothetical protein, conserved 4.3E−05 3.4 Bm1_25670 hypotheticalprotein 4.0E−05 Specific Bm1_41650 hypothetical protein 3.2E−05 4.1Bm1_54890 RNA recognition motif. 3.0E−05 2.0 Bm1_16685 hypotheticalprotein 2.7E−05 2.7 Bm1_40395 Phosphatidylinositol 3- and 4-kinasefamily 2.6E−05 4.3 protein Bm1_00750 RhoGEF domain containing protein1.9E−05 3.4 Bm1_25450 Formin Homology 2 Domain containing 1.8E−05Specific protein Bm1_05305 HYPOTHETICAL PROTEIN, 1.7E−05 SpecificCONSERVED Bm1_42420 Eye-specific diacylglycerol kinase, putative 1.6E−05Specific Bm1_17115 conserved hypothetical protein 9.8E−06 Specific

Summary of the Results

In conclusion, the results detail the proteins found within the majoranatomic fractions of B. malayi, including the intestine, body wall, andreproductive tract. The results suggest that the intestine of adultfilarial worms likely plays an important role in digestion andabsorption, and may have other physiologic functions that have not yetbeen characterized. Further, we have identified vaccine candidates fromthe B. malayi intestine that that could be protective against all majorfilarial pathogens of humans, and which may provide protective efficacyas vaccines against the causative agents of lymphatic filariasis, riverblindness, loiasis as well as heartworm.

Sequences

The sequence identifiers described herein and the sequences set forth inthe following sequence listing correspond to the Accession numbers anddescriptions described in Table 7, below.

TABLE 7 SEQ ID Gene Symbol and NO: Protein Type Description CellAdhesion 1 XP_001899381 Bm1_39630 Immunoglobulin I-set domain containingprotein 2 XP_001892066 Bm1_02820 EGF-like domain containing protein CellSignaling 3 XP_001897556 Bm1_30585 Tyrosine-protein kinaseabl-1.-related 4 XP_001895334 Bm1_19395 Protein kinase domain containingprotein 5 XP_001899110 Bm1_38285 Ser/Thr protein phosphatase familyprotein Chaperone/HSP 6 XP_001894589 Bm1_15660 DnaJ domain containingprotein 7 XP_001895946 Bm1_22450 hemimethylated DNA binding domaincontaining protein Glycosylation/ glucuronidation 8 XP_001900394Bm1_44655 Fukutin.-related 9 XP_001894161 Bm1_13480 UDP-glucoronosyl andUDP-glucosyl transferase family protein Miscellaneous 10 XP_001901384Bm1_49590 CG3054-PA-related 11 XP_001893572 Bm1_10500 AMOP domaincontaining protein 12 XP_001901064 Bm1_48010 EGF-like domain containingprotein Proteases 13 XP_001899113 Bm1_38300 Peptidase family M1containing protein 14 XP_001893672 Bm1_11005 MGC84665 protein- related15 XP_001902078 Bm1_53050 Reprolysin Possible Proteases 16 XP_001891567Bm1_00205 ShTK domain containing protein Protease Inhibitors 17XP_001893428.1 Bm1_09775 “serpin, putative” Hypothetical Proteins 18XP_001901910 Bm1_52210 hypothetical protein 19 XP_001902925 Bm1_57335“Conserved hypothetical protein, putative” 20 XP_001895546 Bm1_20460hypothetical protein 21 XP_001900482 Bm1_45100 hypothetical protein 22XP_001893045 Bm1_07875 CONSERVED HYPOTHETICAL PROTEIN 23 XP_001894967Bm1_17550 hypothetical protein 24 XP_001893039 Bm1_07845 hypotheticalprotein 25 XP_001894908 Bm1_17255 hypothetical protein 26 XP_001895519Bm1_20325 Hypothetical protein- conserved 27 XP_001900708 Bm1_46230hypothetical protein

TABLE A BLAST-P OF INTESTINE ENRICHED NON-MITOCHONDRIAL PROTEINS WITHTRANSMEMBRANE DOMAINS AGAINST W. BANCROFTI, O. VOLVULUS AND H. SAPIENSB. malayi H. sapiens W. bancrofti O. volvulus Protein Type Gene Symbol/Query Query Query Accession No./GI No. Description % Ident* cov.** %Ident* Cov** % Ident* Cov** Acyltransferase Bm1_43465/ Temporarilyassigned gene name protein 40, xx xx 93 290-882  79 50-878 XP_001900154.1/ putative GI: 170590790 Carbohydrate MetabolismBm1_36055/ hexokinase, putative 47 16-437 65 22-438  82 1-439XP_001898658.1/ GI: 170587792 Bm1_52335/UDP-N-acetylglucosamine-dolichyl- 49  2-404 94 1-406 89 1-406XP_001901935.1/ phosphate N- GI: 170594367acetylglucosaminephosphotransferase, putative Catabolism Bm1_48180/Amidase family protein 41 45-303 96 90-324  75 1-373 XP_001901098.1/ GI:170592691 Cell Adhesion Bm1_39630/ Immunoglobulin I-set domaincontaining 28  45-1170 97 628-1171  87 26-1171 XP_001899381.1/ proteinGI:170589239 Bm1_02820/ EGF-like domain containing protein 35 53-206 961-269 82 3-269 XP_001892066.1/ GI:170572325 Cell Signaling Bm1_30585/Tyrosine-protein kinase abl-1.-related 24 212-281  95 10-281  79 10-281 XP_001897556.1/ GI: 170585572 Bm1_19395/ Protein kinase domaincontaining protein 34  15-1280 98 1-681 92  1-1280 XP_001895334.1/GI:170580602 Bm1_38285/ Ser/Thr protein phosphatase family 40 22-287 959-293 79 2-293 XP_001899110.1/ protein GI: 170588697 Bm1_51260/ Innexinfamily protein 29 327-399  83 1-523 64 1-533 XP_001901720.1/ GI:170593937 Bm1_31730/ phosphatidate cytidylyltransferase- 27 56-127 971-128 84 1-118 XP_001897792.1/ related GI:170586046 Bm1_36290/ Putativephosphatidate 50 41-480 94 1-374 85 5-487 XP_001898705.1/cytidylyltransferase, putative GI:170587887 Bm1_43990/ Latrophilinreceptor protein 2, putative 42 354-715  81 59-873  68 32-873 XP_001900260.1/ GI:170591002 Bm1_05960/ Patched family protein 20229-896  96 66-549  73 1-933 XP_001892664.1/ GI:170574085 Bm1_23705/protein C24B5.3, putative 21  8-957 32 12-955  83 2-746 XP_001896198.1/GI:170582592 Bm1_39815/ Ly-6-related protein HOT-2-related 29 18-55  9597-199  89 60-199  XP_001899418.1/ GI:170589313 Bm1_55745/ sulfakininreceptor protein, putative 28  5-333 86 1-299 71 1-398 XP_001902606.1/GI:170596018 Bm1_54240/ Endonuclease/Exonuclease/phosphatase 38  3-29898 1-205 85 1-391 XP_001902307.1/ family protein GI:170595251 Bm1_52975/ER lumen protein retaining 69  1-207 99 1-213 96 1-213 XP_001902063.1/receptor, putative GI:170594623 Bm1_48590/ Low-density lipoprotein 474-46 91 1-154 66 1-154 XP_001901181.1/ receptor domain class AGI:170592857 containing protein Cellular Trafficking Bm1_14235/ SNAREdomain containing 37  9-230 98 1-248 86 1-248 XP_001894314.1/ proteinGI:170578208 Chaperone/HSP Bm1_15660/ DnaJ domain containing 27 25-83591 1-839 77 3-839 XP_001894589.1/ protein GI:170578901 Bm1_22450/hemimethylated DNA 25 34-112 97 29-119  95 29-119  XP_001895946.1/binding domain containing GI:170582031 protein DNA/RNA BindingBm1_41070/ Zinc finger DHHC domain 53 11-262 97 1-445 86 1-444XP_001899675.1/ containing protein 5, GI:170589828 putativeGlycosylation/glucuronidation Bm1_34610/ glycosyl transferase, group 2family 56 80-554 96 1-447 88 1-582 XP_001898369.1/ protein GI:170587206Bm1_44655/ Fukutin.-related 31 138-364  96 1-362 73 1-364XP_001900394.1/ GI:170591272 Bm1_13480/ UDP-glucoronosyl andUDP-glucosyl 27 35-509 95 1-425 29 214-293  XP_001894161.1/ transferasefamily protein GI:170577851 Immunological Bm1_50985/ Complementcomponent C6 precursor.- 32 49-136 98 1-171 72 3-161 XP_001901665.1/related GI:170593827 Miscellaneous Bm1_15480/ Acyltransferase familyprotein 28 295-352  94 44-351  80 17-649  XP_001894556.1/ GI:170578816Bm1_23850/ cDNA sequence BC017158-related 37 38-384 93 163-353  81 1-389XP_001896227.1/ GI:170582654 Bm1_49590/ CG3054-PA-related 28 97-260 811-242 69 1-260 XP_001901384.1/ GI:170593263 Bm1_15855/ D4Ertd196eprotein, putative 56 14-174 94 1-177 81 1-150 XP_001894628.1/GI:170578992 Bm1_10500/ AMOP domain containing protein 26 652-932  98679-1377  92  1-1513 XP_001894628.1/ GI:170578992 Bm1_06760/ zgc: 100814protein-related 39  9-355 95 1-136 75 1-356 XP_001892822.1/ GI:170574457Bm1_41280/ uncharacterized hypothalamus protein 56 50-295 96 1-296 824-296 XP_001899713.1/ HTMP, putative GI:170589904 Bm1_17180/ MiaB-liketRNA modifying enzyme, 56  1-402 97 17-256  90 1-424 XP_001894893.1/archaeal-type family protein GI:170579583 Bm1_48010/ EGF-like domaincontaining protein 36 10-395 91 19-338  66 6-560 XP_001901064.1/GI:170592623 Proteases Bm1_18805/ Papain family cysteine proteasecontaining 34 65-309 68 73-267  52 35-309  XP_001895218.1/ proteinGI:170580338 Bm1_38300/ Peptidase family M1 containing protein 28136-586  90 90-819  67 1-819 XP_001899113.1/ GI:170588703 Bm1_26370/Rhomboid family protein 36 77-347 96 1-377 83 1-377 XP_001896728.1/GI:170583765 Bm1_11005/ MGC84665 protein-related 38 2-98 95 1-96  801-98  XP_001896728.1/ GI:170583765 Bm1_53050/ Reprolysin 32 153-753  9196-845  77 1-839 XP_001902078.1/ GI:170594653 Possible ProteaseBm1_00205/ ShTK domain containing protein 27 54-161 80 142-229  5270-227  XP_001891567.1/ GI:170571016 Protease inhibitor Bm1_09775/serpin, putative 29 26-388 75 84-375  52 29-390  XP_001893428.1/GI:170575897 Sterol Metabolism Bm1_37660/ Oxysterol-binding protein 4537-747 93 312-748  87 57-724  XP_001898982.1/ GI:170588441 StructuralBm1_53475/ Nematode cuticle collagen N-terminal 40 102-274  85 48-288 61 1-291 XP_001902163.1/ domain containing protein GI:170594883Bm1_04695/ Cuticle collagen F09G8.6.-related 40 80-251 96 43-270  891-266 XP_001892417.1/ GI:170573305 Bm1_19730/ Autophagy protein Apg9containing 43 50-655 91 204-814  82 1-814 XP_001895401.1/ proteinGI:170580769 Transporters Bm1_08720/ ABC transporter N-terminus familyprotein 46 65-153 94 1-150 78 1-153 XP_001893214.1/ GI:170575377Bm1_06830/ ABC transporter transmembrane region 40 80-246 92 82-183  601-248 XP_001892835.1/ family protein GI:170574488 Bm1_08185/ Cationtransporter family protein 37 35-439 96 1-444 84 3-444 XP_001893108.1/GI:170575121 Bm1_34425/ Ctr copper transporter family protein 28 63-25589 1-276 69 10-276  XP_001898332.1/ GI:170587131 Bm1_00795/ E1-E2 ATPasefamily protein 49  6-523 96 1-449 87 1-530 XP_001891680.1/ GI:170571314Bm1_42365/ ZIP Zinc transporter family protein 25 17-386 92 1-387 761-387 XP_001899937.1/ GI:170590354 Bm1_40010/ NRAMP-like transporterK11G12.4, 59 44-457 100 228-457  85 34-457  XP_001899457.1/ putativeGI:170589391 Bm1_38955/ Twik family of potassium channels protein 24101-536  93 1-561 96 49-561  XP_001899244.1/ 28, putative GI:170588965Bm1_44770/ TWiK family of potassium channels 26 136-438  98 80-330  871-525 XP_001900417.1/ protein 7, putative GI:170591318 Bm1_24840/ MajorFacilitator Superfamily protein 27  7-477 95 102-493  85 1-493XP_001896428.1/ GI:170583097 Bm1_37140/ Major Facilitator Superfamilyprotein 34 70-221 90 100-739  69 1-739 XP_001898875.1/ GI:170588227Bm1_24985/ cation efflux family protein 26 181-300  97 54-483  8015-483  XP_001896457.1/ GI:170583158 Bm1_46360/ Transmembrane amino acidtransporter 27 29-447 97 32-443  93 1-401 XP_001900734.1/ proteinGI:170591953 Bm1_25200/ Mitochondrial carrier C16C10.1, putative 4712-332 97 1-332 84 1-332 XP_001896499.1/ GI:170583257 Bm1_01695/RE11181p-related 28 14-258 89 1-360 22 1-258 XP_001891855.1/GI:170571765 Bm1_42075/ zgc: 92765, putative 49 11-351 81 1-397 80 1-396XP_001899877.1/ GI:170590234 Bm1_43555/ ABC TRANSPORTER 40  19-1536 9178-1087 71 549-1536  XP_001900172.1/ TRANSMEMBRANE REGION FAMILYGI:170590826 PROTEIN Bm1_37475/ Major Facilitator Superfamily protein 33100-320  93 67-806  76 1-791 XP_001898945.1/ GI:170588367 Bm1_38360/major facilitator superfamily protein 30 41-553 98 31-565  92 31-564 XP_001899125.1/ GI:170588727 Bm1_15490/ ABC transporter family protein45 67-706 98 35-543  81 4-712 XP_001894558.1/ GI:170578824 Bm1_31305/vesicular acetylcholine transporter unc-17, 50 17-440 94 20-528  911-528 XP_001897706.1/ putative GI:170585872 Bm1_42930/ Excitatory aminoacid transporter, putative 59  7-469 98 1-320 92 245-498 XP_001900048.1/ GI:170590576 Bm1_02560/ Sodium/calcium exchanger protein35 29-341 86 156-342  78 1-342 XP_001892014.1/ GI:170572178 Bm1_31865/Probable calcium-binding mitochondrial 45 46-504 93 5-508 86 4-506XP_001897818.1/ carrier F55A11.4, putative GI:170586099 HypotheticalProteins Bm1_04935/ hypothetical protein 47 4-37 28 1-54  65 1-68 XP_001892465.1/ GI:170573424 Bm1_33605/ hypothetical protein 41 42-70 81 1-216 49 58-216  XP_001898167.1/ GI:170586800 Bm1_34095/ hypotheticalprotein 29 15-88  42 55-87  68 1-131 XP_001898267.1/ GI:170587000Bm1_22820/ hypothetical protein 35 30-69  100 1-68  94 1-81 XP_001896020.1/ GI:170582194/ Bm1_26845/ Hypothetical protein 33 77-16492 1-162 79 58-245  XP_001896819.1/ GI:170584006 Bm1_45625/ hypotheticalprotein 38 127-171  90 1-338 76 25-334  XP_001900588.1/ GI:170591660Bm1_52210/ hypothetical protein 29 238-350  97 53-433  85 1-431XP_001901910.1/ GI:170594317 Bm1_02860 hypothetical protein xx xx 3311-31  38 11-31  Bm1_57335/ Conserved hypothetical protein, putative 31114-236  96 1-246 89 1-246 XP_001902925.1/ GI:170596866/ Bm1_25895/hypothetical protein 28 115-179  96 122-325  68 33-325  XP_001896638.1/GI:170583561 Bm1_26820/ hypothetical protein 38 318-378  88 1-317 591-191 XP_001896814.1/ GI:170583994 Bm1_46300/ hypothetical protein,conserved 38 129-154  91 195-387  52 30-387  XP_001900722.1/GI:170591929 Bm1_20460/ hypothetical protein 30 103-147  24 94-147  821-191 XP_001895546.1/ GI:170581122 Bm1_27875/ hypothetical protein 28186-237  91 61-298  76 12-202  XP_001897026.1/ GI:170584478 Bm1_30935/hypothetical protein 39 37-67  82 4-90  27 30-89  XP_001897629.1/GI:170585718 Bm1_53700/ hypothetical protein 37 56-108 92 178-216  561-220 XP_001902205.1/ GI:170594994 Bm1_57235/ hypothetical protein 4039-81  50 45-62  65 1-67  XP_001902905.1/ GI:170596815 Bm1_45100/hypothetical protein 29 330-414  85 118-727  60 75-727  XP_001900482.1/GI:170591448 Bm1_07875/ CONSERVED HYPOTHETICAL 31 84-225 99 142-231  8553-231  XP_001893045.1/ PROTEIN GI:170574981 Bm1_30410/ conservedhypothetical protein 38 152-201  99 112-234  89 105-234  XP_001897520.1/GI:170585498 Bm1_17550/ hypothetical protein 33 63-121 87 77-129  8282-125  XP_001894967.1/ GI:170579749 Bm1_00920/ Hypothetical 30.5 kDaprotein ZK1321.3 in 25  5-168 93 1-176 72 1-176 XP_001891702.1/chromosome II.-related GI:170571368 Bm1_04875/ hypothetical protein xxxx xx xx xx xx XP_001892453.1/ GI:170573392 Bm1_29435/ Hypotheticalprotein 30 23-163 96 23-204  83 23-204  XP_001897329.1/ GI:170585108Bm1_42465/ hypothetical protein 28 85-134 84 1-394 70 1-394XP_001899956.1/ GI:170590392 Bm1_48705/ conserved hypothetical protein23 57-267 97 96-267  74 1-268 XP_001901204.1/ GI:170592903 Bm1_32415/Hypothetical 21.5 kDa protein in SEC15- 52 25-168 93 1-176 90 1-176XP_001897928.1/ SAP4 intergenic region.-related GI:170586322 Bm1_07845/hypothetical protein 28 24-108 89 1-210 64 1-210 XP_001893039.1/GI:170574965 Bm1_15300/ Hypothetical protein xx xx xx xx xx xxXP_001894520.1/ GI:170578730 Bm1_17255/ hypothetical protein 21 93-24687 17-251  78 28-251  XP_001894908.1/ GI:170579615 Bm1_18965/Hypothetical protein 45 46-235 91 1-206 78 1-236 XP_001895250.1/GI:170580404 Bm1_20325/ Hypothetical protein-conserved 37  2-215 961-487 89 1-487 XP_001895519.1/ GI:170581054 Bm1_44010/ hypotheticalprotein 42 3-33 92 91-185  26 74-156  XP_001900264.1/ GI:170591010Bm1_46230/ hypothetical protein 29 184-256  91 24-278  62 1-278XP_001900708.1/ GI:170591901 *% Ident” is the percentage of amino acidswithin the query coverage identical to query sequence. **Query cov.”span of amino acids in the query sequence that aligns with the targetsequence producing significant alignment

TABLE B BLAST P OF THE TABLE A INTESTINE PROTEINS AGAINST L. LOA AND D.IMMITIS. B. malayi Protein Type Gene Symbol/ L. loa D. immitis AccessionNo./GI Query Query No. Description % Ident* Cov** % Ident* Cov**Acyltransferase Bm1_43465/ Temporarily assigned gene name protein 8850-664  84 64-882  XP_001900154.1/ 40, putative GI: 170590790Carbohydrate Metabolism Bm1_36055/ hexokinase, putative 63 22-438  841-439 XP_001898658.1/ GI: 170587792 Bm1_52335/UDP-N-acetylglucosamine-dolichyl- 91 1-406 87 1-388 XP_001901935.1/phosphate N- GI: 170594367 acetylglucosaminephosphotransferase, putativeCatabolism Bm1_48180/ Amidase family protein 78 1-373 74 90-373 XP_001901098.1/ GI: 170592691 Cell Adhesion Bm1_39630/ ImmunoglobulinI-set domain containing 89  1-1171 82  1-1171 XP_001899381.1/ proteinGI: 170589239 Bm1_02820/ EGF-like domain containing protein 89 62-269 79 2-269 XP_001892066.1/ GI: 170572325 Cell Signaling Bm1_30585/Tyrosine-protein kinase abl-1.-related 83 10-281  84 10-281 XP_001897556.1/ GI: 170585572 Bm1_19395/ Protein kinase domaincontaining protein 95  1-1280 93  1-1280 XP_001895334.1/ GI: 170580602Bm1_38285/ Ser/Thr protein phosphatase family protein 88 9-293 79  1-290XP_001899110.1/ GI: 170588697 Bm1_51260/ Innexin family protein 69 1-52364 1-532 XP_001901720.1/ GI: 170593937 Bm1_31730/ phosphatidatecytidylyltransferase-related 93 1-125 87 1-125 XP_001897792.1/ GI:170586046 Bm1_36290/ Putative phosphatidate cytidylyltransferase, 895-487 78 1-487 XP_001898705.1/ putative GI: 170587887 Bm1_43990/Latrophilin receptor protein 2, putative 76 19-841  69 27-873 XP_001900260.1/ GI: 170591002 Bm1_05960/ Patched family protein 87 1-93475 1-933 XP_001892664.1/ GI: 170574085 Bm1_23705/ protein C24B5.3,putative 90 1-934 93 648-959  XP_001896198.1/ GI: 170582592 Bm1_39815/Ly-6-related protein HOT-2-related 89 69-199  89 6-189 XP_001899418.1/GI: 170589313 Bm1_55745/ sulfakinin receptor protein, putative 36 4-33364 139-392  XP_001902606.1/ GI: 170596018 Bm1_54240/Endonuclease/Exonuclease/phosphatase 88 1-256 83 1-391 XP_001902307.1/family protein GI: 170595251 Bm1_52975/ ER lumen protein retainingreceptor, 96 1-213 96 1-209 XP_001902063.1/ putative GI: 170594623Bm1_48590/ Low-density lipoprotein receptor domain 74 4-154 67 9-154XP_001901181.1/ class A containing protein GI: 170592857 CellularTrafficking Bm1_14235/ SNARE domain containing protein 94 1-248 83 1-235XP_001894314.1/ GI: 170578208 Chaperone/HSP Bm1_15660/ DnaJ domaincontaining protein 85 11-839  80 1-839 XP_001894589.1/ GI: 170578901Bm1_22450/ hemimethylated DNA binding domain 97 29-119  91 28-119 XP_001895946.1/ containing protein GI: 170582031 DNA/RNA BindingBm1_41070/ Zinc finger DHHC domain containing 90 28-445  88 1-445XP_001899675.1/ protein 5, putative GI: 170589828 Glycosylation/glucuronidation Bm1_34610/ glycosyl transferase, group 2 family protein89 1-435 88 1-582 XP_001898369.1/ GI: 170587206 Bm1_44655/Fukutin.-related 85 1-364 75 1-364 XP_001900394.1/ GI: 170591272Bm1_13480/ UDP-glucoronosyl and UDP-glucosyl 81 1-423 72 155-502 XP_001894161.1/ transferase family protein GI: 170577851 ImmunologicalBm1_50985/ Complement component C6 precursor.- 61 2-166 75 3-171XP_001901665.1/ related GI: 170593827 Miscellaneous Bm1_15480/Acyltransferase family protein 86 266-467  76 49-638  XP_001894556.1/GI: 170578816 Bm1_23850/ cDNA sequence BC017158-related 82 1-389 7221-389  XP_001896227.1/ GI: 170582654 Bm1_49590/ CG3054-PA-related 631-262 67 1-254 XP_001901384.1/ GI: 170593263 Bm1_15855/ D4Ertd196eprotein, putative 86 1-177 81 1-151 XP_001894628.1/ GI: 170578992Bm1_10500/ AMOP domain containing protein 94  1-1513 90 609-1513 XP_001894628.1/ GI: 170578992 Bm1_06760/ zgc: 100814 protein-related 841-357 71 1-356 XP_001892822.1/ GI: 170574457 Bm1_41280/ uncharacterizedhypothalamus protein 81 1-296 80 1-296 XP_001899713.1/ HTMP, putativeGI: 170589904 Bm1_17180/ MiaB-like tRNA modifying enzyme, 94 1-390 951-265 XP_001894893.1/ archaeal-type family protein GI: 170579583Bm1_48010/ EGF-like domain containing protein 74 6-556 64 6-546XP_001901064.1/ GI: 170592623 Proteases Bm1_18805/ Papain familycysteine protease 56 134-313  41 1-314 XP_001895218.1/ containingprotein GI: 170580338 Bm1_38300/ Peptidase family M1 containing protein74 1-819 70 1-819 XP_001899113.1/ GI: 170588703 Bm1_26370/ Rhomboidfamily protein 83 1-377 85 17-377  XP_001896728.1/ GI: 170583765Bm1_11005/ MGC84665 protein-related 91 1-98  90 1-40  XP_001896728.1/GI: 170583765 Bm1_53050/ Reprolysin 79 1-839 77 1-843 XP_001902078.1/GI: 170594653 Possible Protease Bm1_00205/ ShTK domain containingprotein 46 126-264  55 110-264  XP_001891567.1/ GI: 170571016 Proteaseinhibitor Bm1_09775/ serpin, putative 54 1-391 52 1-391 XP_001893428.1/GI: 170575897 Sterol Sterol Metabolism Metabolism Bm1_37660/Oxysterol-binding protein 82 57-753  86 57-753  XP_001898982.1/ GI:170588441 Structural Structural BBm1_53475/ Nematode cuticle collagenN-terminal 64 1-281 55 46-291  XP_001902163.1/ domain containing proteinGI: 170594883 Bm1_04695/ Cuticle collagen F09G8.6.-related 89 1-270 861-269 XP_001892417.1/ GI: 170573305 Bm1_19730/ Autophagy protein Apg9containing protein 86 1-814 81 1-814 XP_001895401.1/ GI: 170580769Transporters Bm1_08720/ ABC transporter N-terminus family protein 841-153 72 1-153 XP_001893214.1/ GI: 170575377 Bm1_06830/ ABC transportertransmembrane region 81 82-248  62 1-244 XP_001892835.1/ family proteinGI: 170574488 Bm1_08185/ Cation transporter family protein 57 32-345  5967-345  XP_001893108.1/ GI: 170575121 Bm1_34425/ Ctr copper transporterfamily protein 74 2-276 73 1-276 XP_001898332.1/ GI: 170587131Bm1_00795/ E1-E2 ATPase family protein 91 45-531  85 1-529XP_001891680.1/ GI: 170571314 Bm1_42365/ ZIP Zinc transporter familyprotein 81 28-387  43 17-386  XP_001899937.1/ GI: 170590354 Bm1_40010/NRAMP-like transporter K11G12.4, putative 91 1-456 83 1-457XP_001899457.1/ GI: 170589391 Bm1_38955/ Twik family of potassiumchannels protein 92 1-561 91 1-561 XP_001899244.1/ 28, putative GI:170588965 Bm1_44770/ TWiK family of potassium channels proteinXP_001900417.1/ 7, putative 94 1-525 91 1-525 GI: 170591318 Bm1_24840/Major Facilitator Superfamily protein 87 1-418 86 1-493 XP_001896428.1/GI: 170583097 Bm1_37140/ Major Facilitator Superfamily protein 75 1-73871 1-739 XP_001898875.1/ GI: 170588227 Bm1_24985/ cation efflux familyprotein 85 1-483 81 1-483 XP_001896457.1/ GI: 170583158 Bm1_46360/Transmembrane amino acid transporter 86 1-443 91 1-439 XP_001900734.1/protein GI: 170591953 Bm1_25200/ Mitochondrial carrier C16C10.1,putative 89 1-332 83 1-332 XP_001896499.1/ GI: 170583257 Bm1_01695/RE11181p-related 26 4-105 23 4-258 XP_001891855.1/ GI: 170571765Bm1_42075/ zgc: 92765, putative 84 1-396 74 1-396 XP_001899877.1/ GI:170590234 Bm1_43555/ ABC TRANSPORTER 71 12-996  71 14-1423XP_001900172.1/ TRANSMEMBRANE REGION GI: 170590826 FAMILY PROTEINBm1_37475/ Major Facilitator Superfamily protein 82 1-792 78 113-795 XP_001898945.1/ GI: 170588367 Bm1_38360/ major facilitator superfamilyprotein 93 31-565  91 21-564  XP_001899125.1/ GI: 170588727 Bm1_15490/ABC transporter family protein 89 4-713 77 7-638 XP_001894558.1/ GI:170578824 Bm1_31305/ vesicular acetylcholine transporter unc- 92 1-52887 1-528 XP_001897706.1/ 17, putative GI: 170585872 Bm1_42930/Excitatory amino acid transporter, 92 1-498 93 1-499 XP_001900048.1/putative GI: 170590576 Bm1_02560/ Sodium/calcium exchanger protein 791-342 71 1-314 XP_001892014.1/ GI: 170572178 Bm1_31865/ Probablecalcium-binding 82 8-501 86 4-508 XP_001897818.1/ mitochondrial carrierF55A11.4, GI: 170586099 putative Hypothetical Proteins Bm1_04935/hypothetical protein 72 1-111 66 1-112 XP_001892465.1/ GI: 170573424Bm1_33605/ hypothetical protein 58 111-216  22 19-188  XP_001898167.1/GI: 170586800 Bm1_34095/ hypothetical protein 81 1-131 71 1-129XP_001898267.1/ GI: 170587000 Bm1_22820/ hypothetical protein 98 1-81 96 1-81  XP_001896020.1/ GI: 170582194/ Bm1_26845/ Hypothetical protein81 63-162  72 1-245 XP_001896819.1/ GI: 170584006 Bm1_45625/hypothetical protein 82 1-332 76 1-332 XP_001900588.1/ GI: 170591660Bm1_52210/ hypothetical protein 87 15-432  84 1-430 XP_001901910.1/ GI:170594317 Bm1_02860 hypothetical protein 33 11-31  33 11-31  Bm1_57335/Conserved hypothetical protein, putative 91 1-246 87 1-246XP_001902925.1/ GI: 170596866/ Bm1_25895/ hypothetical protein 7431-325  67 24-325  XP_001896638.1/ GI: 170583561 Bm1_26820/ hypotheticalprotein 77 1-317 72 99-317  XP_001896814.1/ GI: 170583994 Bm1_46300/hypothetical protein, conserved 75 1-191 56 18-380  XP_001900722.1/ GI:170591929 Bm1_20460/ hypothetical protein 86 1-191 85 1-191XP_001895546.1/ GI: 170581122 Bm1_27875/ hypothetical protein 79 12-202 77 185-298  XP_001897026.1/ GI: 170584478 Bm1_30935/ hypotheticalprotein 64 9-90  28 17-70  XP_001897629.1/ GI: 170585718 Bm1_53700/hypothetical protein 57 53-236  62 2-103 XP_001902205.1/ GI: 170594994Bm1_57235/ hypothetical protein 85 10-67  74 1-68  XP_001902905.1/ GI:170596815 Bm1_45100/ hypothetical protein 67 81-696  49 271-727 XP_001900482.1/ GI: 170591448 Bm1_07875/ CONSERVED HYPOTHETICAL 9042-231  90 41-231  XP_001893045.1/ PROTEIN GI: 170574981 Bm1_30410/conserved hypothetical protein 90 1-234 94 154-234  XP_001897520.1/ GI:170585498 Bm1_17550/ hypothetical protein 77 77-129  86 82-125 XP_001894967.1/ GI: 170579749 Bm1_00920/ Hypothetical 30.5 kDa proteinZK1321.3 in 75 1-174 75 1-176 XP_001891702.1/ chromosome II.-related GI:170571368 Bm1_04875/ hypothetical protein xx xx xx xx XP_001892453.1/GI: 170573392 Bm1_29435/ Hypothetical protein 89 23-204  81 23-204 XP_001897329.1/ GI: 170585108 Bm1_42465/ hypothetical protein 25 14-387 25 14-385  XP_001899956.1/ GI: 170590392 Bm1_48705/ conservedhypothetical protein 81 1-268 72 1-269 XP_001901204.1/ GI: 170592903Bm1_32415/ Hypothetical 21.5 kDa protein in SEC15- 93 1-176 91 1-176XP_001897928.1/ SAP4 intergenic region.-related GI: 170586322 Bm1_07845/hypothetical protein 75 1-210 72 60-210 XP_001893039.1/ GI: 170574965Bm1_15300/ Hypothetical protein xx xx xx xx XP_001894520.1/ GI:170578730 Bm1_17255/ hypothetical protein 73 13-251  75 28-251 XP_001894908.1/ GI: 170579615 Bm1_18965/ Hypothetical protein 85 1-23677 1-239 XP_001895250.1/ GI: 170580404 Bm1_20325/ Hypotheticalprotein-conserved 91 1-487 89 1-485 XP_001895519.1/ GI: 170581054Bm1_44010/ hypothetical protein 72 9-185 32 61-129  XP_001900264.1/ GI:170591010 Bm1_46230/ hypothetical protein 65 18-278  31 161-281 XP_001900708.1/ GI: 170591901 *% Ident” is the percentage of amino acidswithin the query coverage identical to query sequence. **Query cov.”span of amino acids in the query sequence that aligns with the targetsequence producing significant alignment

TABLE C SELECTED PROTEINS FROM TABLES A AND B CONTAINING 1-2TRANSMEMBRANE DOMAINS, A SIGNIFICANT NON-CYTOPLASMIC PORTION, >75%HOMOLOGY TO EITHER W. BANCROFTI OR O. VOLVULUS AND <40% HOMOLOGY TOHUMANS. H. sapiens W. bancrofti O. volvulus L. loa D. immitis SEQ IDNO:/ Query Query Query Query Query Gene Symbol % Ident* cov**. % Ident*Cov** % Ident* Cov** % Ident* Cov** % Ident* Cov** 1/Bm1_39630/ 28 45-1170 97 628-1171 87 26-1171 89  1-1171 82  1-1171 XP_001899381.1/GI:170589239 2/Bm1_02820/ 35 53-206 96  1-269 82 3-269 89 62-269  792-269 XP_001892066.1/ GI:170572325 3/Bm1_30585/ 24 212-281  95 10-281 7910-281  83 10-281  84 10-281  XP_001897556.1/ GI:170585572 4/Bm1_19395/34  15-1280 98  1-681 92  1-1280 95  1-1280 93  1-1280 XP_001895334.1/GI:170580602 5/Bm1_38285/ 40 22-287 95  9-293 79 2-293 88 9-293 79 1-290XP_001899110.1/ GI:170588697 6/Bm1_15660/ 27 25-835 91  1-839 77 3-83985 11-839  80 1-839 XP_001894589.1/ GI:170578901 7/Bm1_22450/ 25 34-11297 29-119 95 29-119  97 29-119  91 28-119  XP_001895946.1/ GI:1705820318/Bm1_44655/ 31 138-364  96  1-362 73 1-364 85 1-364 75 1-364XP_001900394.1/ GI:170591272 9/Bm1_13480/ 27 35-509 95  1-425 29214-293  81 1-423 72 155-502  XP_001894161.1/ GI:170577851 10/Bm1_49590/28 97-260 81  1-242 69 1-260 63 1-262 67 1-254 XP_001901384.1/GI:170593263 11/Bm1_10500/ 26 652-932  98 679-1377 92  1-1513 94  1-151390 609-1513  XP_001893572.1/ GI:170576299/ 12/Bm1_48010/ 36 10-395 9119-338 66 6-560 74 6-556 64 6-546 XP_001901064.1 GI:170592623/13/Bm1_38300/ 28 136-586  90 90-819 67 1-819 74 1-819 70 1-819XP_001899113.1/ GI:170588703 14/Bm1_11005/ 38 2-98 95 1-96 80 1-98  911-98  90 1-40  XP_001893672.1/ GI:170576544 15/Bm1_53050/ 32 153-753  9196-845 77 1-839 79 1-839 77 1-843 XP_001902078.1/ GI:17059465316/Bm1_00205/ 27 54-161 80 142-229  52 70-227  46 126-264  55 110-264 XP_001891567.1/ GI:170571016 17/Bm1_09775/ 29 26-388 75 84-375 5229-390  54 1-391 52 1-391 XP_001893428.1/ GI:170575897 18/Bm1_52210/ 29238-350  97 53-433 85 1-431 87 15-432  84 1-430 XP_001901910.1/GI:170594317 19/Bm1_57335/ 31 114-236  96  1-246 89 1-246 91 1-246 871-246 XP_001902925.1/ GI:170596866 20/Bm1_20460/ 30 103-147  24 94-14782 1-191 86 1-191 85 1-191 XP_001895546.1/ GI:170581122 21/Bm1_45100/ 29330-414  85 118-727  60 75-727  67 81-696  49 271-727  XP_001900482.1/GI:170591448 22/Bm1_07875/ 31 84-225 99 142-231  85 53-231  90 42-231 90 41-231  XP_001893045.1/ GI:170574981 23/Bm1_17550/ 33 63-121 8777-129 82 82-125  77 77-129  86 82-125  XP_001894967.1/ GI:17057974924/Bm1_07845/ 28 24-108 89  1-210 64 1-210 75 1-210 72 60-210 XP_001893039.1/ GI:170574965 25/Bm1_17255/ 21 93-246 87 17-251 7828-251  73 13-251  75 28-251  XP_001894908.1/ GI:170579615 26/Bm1_20325/37  2-215 96  1-487 89 1-487 91 1-487 89 1-485 XP_001895519.1/GI:170581054 27/Bm1_46230/ 29 184-256  91 24-278 62 1-278 65 18-278  31161-281  XP_001900708.1/ GI:170591901 *% Ident” is the percentage ofamino acids within the query coverage identical to query sequence.**Query cov.” span of amino acids in the query sequence that aligns withthe target sequence producing significant alignment

TABLE D NORMALIZED SPECTRAL ABUNDANCE FACTOR (NSAF) AND NSAF ENRICHMENTNSAF enrichment* NSAF Reproductive Abundance** Non-cytoplasmicTransmembrane SEQ ID NO. Intestine Body Wall Tract Intestine domain^(a)(TMHMM)^(b) 1/Bm1_39630/ 3.84 0.26 0 2.20E−05 19-1120 1 XP_001899381.1/GI:170589239 2/Bm1_02820/ 9999 0 0 1.30E−04 1-225 1 XP_001892066.1/GI:170572325 3/Bm1_30585/ 4.42 0 0.23 8.40E−05 19-135  1 XP_001897556.1/GI:170585572 4/Bm1_19395/ 3.42 0.14 0.12 3.30E−05 1-942 1XP_001895334.1/ GI:170580602 5/Bm1_38285/ 7.68 0.13 0 1.80E−04 41-293  1XP_001899110.1/ GI:170588697 6/Bm1_15660/ 10.31 0 0.1 7.20E−05 18-220  1XP_001894589.1/ GI:170578901 7/Bm1_22450/ 2.74 0.17 0.14 2.70E−04 1-1251 XP_001895946.1/ GI:170582031 8/Bm1_44655/ 2.95 0 0.34 4.70E−05 28-364 1 XP_001900394.1/ GI:170591272 9/Bm1_13480/ 28.16 0.04 0 7.40E−04 1-4861 XP_001894161.1/ GI:170577851 10/Bm1_49590/ 2.95 0 0.34 6.50E−05 1-51,101-265 2 XP_001901384.1/ GI:170593263 11/Bm1_10500/ 5.99 0.01 0.156.10E−04 23-1322 1 XP_001893572.1/ GI:170576299/ 12/Bm1_48010/ 2.03 0.380.06 2.50E−04 1-430 1 XP_001901064.1 GI:170592623/ 13/Bm1_38300/ 9999 00 1.60E−05 81-1061 1 XP_001899113.1/ GI:170588703 14/Bm1_11005/ 2.740.17 0.14 2.00E−04 1-16, 76-169  1 XP_001893672.1/ GI:17057654415/Bm1_53050/ 2.95 0 0.34 6.10E−04 1-607 1 XP_001902078.1/ GI:17059465316/Bm1_00205/ 3.42 0.23 0.04 4.200+00 26-264  1 XP_001891567.1/GI:170571016 17/Bm1_09775/ 2.56 0.39 0 4.70E−05 17-391  1XP_001893428.1/ GI:170575897 18/Bm1_52210/ 9999 0 0 4.00E−05 1-369 1XP_001901910.1/ GI:170594317 19/Bm1_57335/ 9999 0 0 7.00E−05 29-211  2XP_001902925.1/ GI:170596866 20/Bm1_20460/ 2.95 0 0.34 8.00E−05 143-147 2 XP_001895546.1/ GI:170581122 21/Bm1_45100/ 5.12 0.2 0 4.70E−05285-727  1 XP_001900482.1/ GI:170591448 22/Bm1_07875/ 4.42 0 0.231.10E−04 1-56.  1 XP_001893045.1/ GI:170574981 23/Bm1_17550/ 2.56 0.39 01.30E−04 1-61.  1 XP_001894967.1/ GI:170579749 24/Bm1_07845/ 2.09 0.150.24 4.90E−04 53-210  1 XP_001893039.1/ GI:170574965 25/Bm1_17255/ 3.640.2 0.05 3.80E−04 36-251  1 XP_001894908.1/ GI:170579615 26/Bm1_20325/3.36 0.12 0.14 3.00E−04 1-194 1 XP_001895519.1/ GI:17058105427/Bm1_46230/ 2.21 0 0.45 1.80E−04 121-289  1 XP_001900708.1/GI:170591901 *Calculated as described in the Examples **Calculated asdescribed in the Examples ^(a)Non-cytoplasmic domain refers to the spanof amino acids predicted to be non-cytoplasmic as described in theExamples ^(b)Transmembrane refers to number of transmembrane domains aspredicted by a membrane protein topology prediction method TMHMM asdescribed in the Examples.

What is claimed is:
 1. An immunogenic composition comprising: at leasttwo isolated polypeptides or immunogenic fragments thereof and anadjuvant, wherein the at least two isolated polypeptides comprise afirst polypeptide comprising the amino acid sequence of SEP ID NO: 1 anda second polypeptide comprising the amino acid sequence of SEP ID NO: 9and the immunogenic fragments thereof comprise at least 30 contiguousamino acid sequences from the amino acid sequence of SEQ ID NO:1 and atleast 30 contiguous amino add sequences from the amino acid sequence ofSEQ ID NO:
 9. 2. The immunogenic composition as in claim 1, furthercomprising one or more polypeptides having an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2-7, 11, 14, 15, 18,19, 22, 23 and
 26. 3. The immunogenic composition as in claim 1, whereinthe immunogenic composition consists essentially of five isolatedpolypeptides and the adjuvant.
 4. The immunogenic composition as inclaim 1, wherein the immunogenic composition is a vaccine.
 5. Theimmunogenic composition as in claim 1, wherein the adjuvant is Freund'scomplete adjuvant.
 6. A method for treating a filarial diseasecomprising administering an effective amount of a vaccine comprising theimmunogenic composition of claim
 1. 7. The method of claim 6, whereinthe filarial disease is selected from the group consisting of lymphaticfilariasis, river blindness, loiasis and heartworm.
 8. The method ofclaim 6, wherein the subject is a human.
 9. The method of claim 6,wherein the filarial disease is heartworm.
 10. The method of claim 6,wherein the subject is a dog.
 11. The method of claim 6, wherein thevaccine is subcutaneously, intradermally, orally, or nasallyadministered.